Prevalence of Neutralizing Antibodies to Japanese Encephalitis Virus among High-Risk Age Groups in South Korea, 2010

<div><p>After an extensive vaccination policy, Japanese encephalitis (JE) was nearly eliminated since the mid-1980s in South Korea. Vaccination in children shifted the affected age of JE patients from children to adults. However, an abrupt increase in JE cases occurred in 2010, and this trend has continued. The present study aimed to investigate the prevalence of neutralizing antibodies to the JE virus (JEV) among high-risk age groups (≥40 years) in South Korea. A plaque reduction neutralization test was conducted to evaluate the prevalence of neutralizing antibodies to JEV in 945 subjects within four age groups (30–39, 40–49, 50–59, and 60–69 years) in 10 provinces. Of the 945 enrolled subjects, 927 (98.1%) exhibited antibodies against JEV. No significant differences were found in the prevalence of neutralizing antibodies according to sex, age, or occupation. However, there were significant differences in the plaque reduction rate according to age and occupation; oldest age group had a higher reduction rate, and subjects who were employed in agriculture or forestry also had a higher value than the other occupations. We also found that three provinces (Gangwon, Jeonnam, and Gyeongnam) had a relatively lower plaque reduction rate than the other locations. In addition, enzyme-linked immunosorbent assays were conducted to determine recent viral infections and 12 (2.2%) subjects were found to have been recently infected by the virus. In conclusion, the present study clearly indicated that the prevalence of neutralizing antibodies has been maintained at very high levels among adult age groups owing to vaccination or natural infections, or both. In the future, serosurveillance should be conducted periodically using more representative samples to better understand the population-level immunity to JE in South Korea.</p></div

Study areas and distribution of study subjects.

<p>Human serum samples were obtained from the National Biobank of Korea. Using simple random sampling, 945 subjects were selected within four age groups from 10 provinces. The number of subjects per area is provided in the rounded rectangle. The number of patients with Japanese encephalitis in 2010 is provided in the black squares. In addition, the incidence rates per 100,000 persons in each province are provided in parenthesis. SU, Seoul; GG, Gyeonggi; CB, Chungbuk; CN, Chungnam; JB, Jeonbuk; JN, Jeonnam; GN, Gyeongnam; GB, Gyeongbuk; GW, Gangwon; JJ, Jeju.</p

A Novel Immunochromatographic Test Applied to a Serological Survey of Japanese Encephalitis Virus on Pig Farms in Korea

<div><p>Among vertebrate species, pigs are a major amplifying host of Japanese encephalitis virus (JEV) and measuring their seroconversion is a reliable indicator of virus activity. Traditionally, the hemagglutination inhibition test has been used for serological testing in pigs; however, it has several limitations and, thus, a more efficient and reliable replacement test is required. In this study, we developed a new immunochromatographic test for detecting antibodies to JEV in pig serum within 15 min. Specifically, the domain III region of the JEV envelope protein was successfully expressed in soluble form and used for developing the immunochromatographic test. The test was then applied to the surveillance of Japanese encephalitis (JE) in Korea. We found that our immunochromatographic test had good sensitivity (84.8%) and specificity (97.7%) when compared with an immunofluorescence assay used as a reference test. During the surveillance of JE in Korea in 2012, the new immunochromatographic test was used to test the sera of 1,926 slaughtered pigs from eight provinces, and 228 pigs (11.8%) were found to be JEV-positive. Based on these results, we also produced an activity map of JEV, which marked the locations of pig farms in Korea that tested positive for the virus. Thus, the immunochromatographic test reported here provides a convenient and effective tool for real-time monitoring of JEV activity in pigs.</p></div

The activity map of Japanese encephalitis virus in Korea in 2012.

<p>The locations of pig farms where at least one pig tested positive are marked as closed circles according to their latitude and longitude. CB, Chungbuk province; CN, Chungnam province; JB, Jeonbuk province; JN, Jeonnam province; GN, Gyeongnam province; GB, Gyeongbuk province; GW, Gangwon province; JJ, Jeju province.</p

Purification of the domain III subunit of the envelope (E) protein and western blot analysis.

<p>(A) Purification of the E protein from the Japanese encephalitis virus (JEV) and (B) western blot analysis using anti-JEV sera. The soluble envelope protein (domain III region) was purified from 50 mL culture using a nickel column. The samples were resolved by 12% gel electrophoresis and transferred onto the PVDF membrane. The blot was probed with JEV-positive sera collected from Japanese encephalitis patients and developed with enhanced chemiluminescent detection reagents. Lanes: M, molecular weight markers; 1, negative control (non-induced cell); 2, whole lysate of the induced cell culture; 3, purified protein fraction from the whole lysate. The arrow on the blot indicates the target protein of size 28.0kDa. The upper bands on lanes 1 and 2 are non-specific and not detected within the purified fraction (lane 3).</p

The immunochromatographic test.

<p>(A) Schematic diagram of the immunochromatographic test and (B) typical results obtained from testing pig serum. Labeled parts: 1, sample pad; 2, gold conjugation pad; 3, test line; 4, control line; 5, absorbance pad; 6, nitrocellulose membrane; 7, plastic backing.</p

Expression of the envelope (E) protein of Japanese encephalitis virus (JEV).

<p>(A) Cloning strategy for the expression of the E protein of JEV and (B) gel electrophoresis of the expressed protein. Three different fragments of the E gene from JEV were cloned into a pBAD/D-TOPO vector and over-expressed in <i>Escherichia coli</i> cells. The supernatant and pellet of lysed cells were analyzed by gel electrophoresis. Lanes: M, molecular weight markers; N, negative control (non-induced cell); S, soluble fraction (supernatant); I, insoluble fraction (pellet).</p

Primers used for cDNA synthesis and amplification of the envelope (E) gene of Japanese encephalitis virus.

<p>^aThe nucleotide position was based on the E gene of the Nakayama strain (GenBank U70413). The sequence of CACC at the 5' end of the primer was added for directional cloning according to the manufacturer's instructions.</p><p>Primers used for cDNA synthesis and amplification of the envelope (E) gene of Japanese encephalitis virus.</p