japanese encephalitis virus ( jev ) is a mosquito - borne microorganism that causes encephalitis in humans and reproductive failure in pregnant sows .
jev has been recently recognized as a reemerging pathogen as it has expanded into new territories such as the torres strait and australia [ 5,10,18 - 20 ] .
based on a sequence analysis of the c / prm and e genes , jev has been classified into five genotypes ; their relationships have been characterized .
jev genotype 1 , which was distributed in limited regions such as thailand and cambodia before the 1990s , has expanded into northeastern asian countries including vietnam , china , japan , and korea .
although the exact mechanisms for the expansion of this genotype are unknown , several factors such as bird migration , wind - blown mosquitoes , and the transport of animals infected with jev have been suggested .
additionally , climate changes caused by recent global warming and extreme weather patterns may have significantly impacted the transmission of vector - borne diseases such as jev , west nile virus ( wnv ) , and tick - borne encephalitis virus ( tbev ) .
this is because rapid weather and climate changes can directly or indirectly affect migratory birds and mosquitoes .
sero - epidemiological studies are critical for predicting potentially important viral disease outbreaks and preventing the introduction of new jev genotypes into korea .
recently , saito et al .   suggested that wild ducks captured in hokkaido , japan can transmit vector - borne viruses into new territories .
migratory birds may serve as viral reservoirs or amplifying hosts , but they do not develop clinical symptoms . although migrating wild birds may be a major jev vector , no epidemiological survey of jev , which could provide valuable information for establishing control measures to prevent jev outbreaks in swine and humans , has been properly conducted for wild birds in korea .
thus , we performed a serological survey to determine the prevalence of antibodies against jev in wild birds captured on the korean peninsula .
a total of 1,316 blood samples were collected from wild birds in 16 locations of six provinces ( gyeonggi - do ; 3 site , gyeongsangnam - do ; 1 site , jeollanam - do ; 4 sites , jeollabuk - do ; 3 sites , chungcheongnam - do ; 3 sites , chungcheongbuk - do ; 2 sites ) of korea between april 2007 and december 2009 for our seroprevalence study .
all wild birds were lured by rice seed and captured using cannon or mist net .
blood sample from wing vein of each bird using sterile syringe of 3 ml was taken .
the nine species of wild birds tested in this study were oceanodroma castro ( four birds ) , anas formosa ( seven ) , anas penelope ( 20 ) , fulica atra ( 30 ) , anas acuta ( 89 ) , anas crecca ( 154 ) , anas platyrhynchos ( 214 ) , aix galericulata ( 310 ) , and anas poecilorhyncha ( 488 ) .
clotted blood samples were separated by 3,000 g , and the sera were stored at -20 until use . before performing the hi test ,
the kv1899 ( genotype 1 strain ) strain of jev was used as the positive antigen for the hi test .
this strain was isolated from korean pig blood in 1999 and has been passed nine times in vero cells after isolation . to estimate the jev antibody prevalence in the wild bird sera ,
an hi test was performed in 96-well microplates ( corning , usa ) using slightly modified standard methods .
a sucrose - acetone extraction method was used to prepare viral antigens from the brains of suckling mice infected with the korean isolate of jev strain kv1899 .
briefly , 10 l of the serum samples collected from wild birds and 50 l of 4% bovine albumin were mixed with 40 l of 25% kaolin ( sigma , usa ) and incubated for 30 min to remove non - specific inhibitors at room temperature . after mixing ,
the clear supernatant was then mixed with 5 l of packed goose erythrocytes to remove any natural agglutinins . after incubating at 37 for 1 h
, the treated sera were separated from the goose erythrocytes by 3,000 g at 4. the sera ( 25 l ) were diluted two - fold from 1 : 20 to 1 : 10,240 in round - bottom 96-well microplates ( corning , usa ) and incubated with 8 hemagglutinating antigen ( ha ) units of jev . after incubating at 37 for 1 h
, 50 l of 0.33% goose erythrocytes were added and the microplates were incubated for 30 min at 37. to confirm the test reliability , porcine control sera positive and negative for jev were used in all hi tests .
hi titer was expressed as the reciprocal of the highest dilution of serum showing complete hemagglutination inhibition .
jev prevalence was estimated as the number of positive samples compared to the number of samples tested and expressed as a percentage . a 95% confidence interval ( ci )
additionally , each titer was classified according to bird species , time ( year ) , and place ( capture location ) .
a total of 1,316 blood samples were collected from wild birds in 16 locations of six provinces ( gyeonggi - do ; 3 site , gyeongsangnam - do ; 1 site , jeollanam - do ; 4 sites , jeollabuk - do ; 3 sites , chungcheongnam - do ; 3 sites , chungcheongbuk - do ; 2 sites ) of korea between april 2007 and december 2009 for our seroprevalence study .
all wild birds were lured by rice seed and captured using cannon or mist net .
blood sample from wing vein of each bird using sterile syringe of 3 ml was taken .
the nine species of wild birds tested in this study were oceanodroma castro ( four birds ) , anas formosa ( seven ) , anas penelope ( 20 ) , fulica atra ( 30 ) , anas acuta ( 89 ) , anas crecca ( 154 ) , anas platyrhynchos ( 214 ) , aix galericulata ( 310 ) , and anas poecilorhyncha ( 488 ) .
clotted blood samples were separated by 3,000 g , and the sera were stored at -20 until use .
before performing the hi test , the sera were inactivated by incubating at 56 for 30 min .
the kv1899 ( genotype 1 strain ) strain of jev was used as the positive antigen for the hi test .
this strain was isolated from korean pig blood in 1999 and has been passed nine times in vero cells after isolation . to estimate the jev antibody prevalence in the wild bird sera ,
an hi test was performed in 96-well microplates ( corning , usa ) using slightly modified standard methods .
a sucrose - acetone extraction method was used to prepare viral antigens from the brains of suckling mice infected with the korean isolate of jev strain kv1899 .
briefly , 10 l of the serum samples collected from wild birds and 50 l of 4% bovine albumin were mixed with 40 l of 25% kaolin ( sigma , usa ) and incubated for 30 min to remove non - specific inhibitors at room temperature . after mixing ,
the clear supernatant was then mixed with 5 l of packed goose erythrocytes to remove any natural agglutinins . after incubating at 37 for 1 h
, the treated sera were separated from the goose erythrocytes by 3,000 g at 4. the sera ( 25 l ) were diluted two - fold from 1 : 20 to 1 : 10,240 in round - bottom 96-well microplates ( corning , usa ) and incubated with 8 hemagglutinating antigen ( ha ) units of jev . after incubating at 37 for 1 h ,
50 l of 0.33% goose erythrocytes were added and the microplates were incubated for 30 min at 37. to confirm the test reliability , porcine control sera positive and negative for jev were used in all hi tests .
hi titer was expressed as the reciprocal of the highest dilution of serum showing complete hemagglutination inhibition .
jev prevalence was estimated as the number of positive samples compared to the number of samples tested and expressed as a percentage . a 95% confidence interval ( ci )
additionally , each titer was classified according to bird species , time ( year ) , and place ( capture location ) .
jev seroprevalence as determined by our study is shown in tables 1~3 , and figs . 1 and 2
. the overall prevalence of antibodies against jev was 86.7% ( 95% ci , 84.7~88.5% ) for the 1,316 serum samples consisting of nine wild bird species captured in the korean peninsula .
prevalence according to year was 68.2% ( 189 positives/277 tested ; 95% ci , 62.4~73.7% ) for 2007 , 86.4% ( 367/425 ; 95% ci , 82.7~89.5% ) for 2008 , and 95.3% ( 585/614 ; 95% ci , 93.3~96.8% ) for 2009 ( fig .
the regional seroprevalence of positive hi titers  1 : 20 ranged from 82.9 to 100% .
compared to other provinces , the gyeongsangnam - do showed the highest prevalence ( 100% ) ; however , no significant difference in regional prevalence was found .
the regional seroprevalences were 100% ( 80/80 ) in gyeongsangnam - do , 93.0% ( 80/86 ; 95% ci , 85.4~97.4% ) in jeollanam - do , 91.1% ( 216/237 ; 95% ci , 86.8~94.4% ) in jeollabuk - do , 84.7% ( 210/248 ; 95% ci , 79.6~88.9% ) in gyeonggi - do , 84.6% ( 176/208 ; 95% ci , 79.0~89.2% ) in chungcheongbuk - do , and 82.9% ( 379/457 ; 95% ci , 79.2~86.3% ) in chungcheongnam - do .
seroprevalence according to wild bird species were 100% ( 4/4 ; 7/7 ; 89/89 ) in oceanodroma castro , anas formosa , and anas acuta ( winter visitor ) ; 96.7% ( 29/30 ; 95% ci , 82.8~99.9% ) in fulica atra , 90.8% ( 443/488 ; 95% ci , 87.9~93.2% ) in anas poecilorhyncha ( migratory breeder ) , 87.9% ( 188/214 ; 95% ci , 82.7~91.9% ) in anas platyrhynchos , 80% ( 16/20 ; 95% ci , 56.3~94.3% ) in anas penelope ( winter visitor ) , 78.7% in aix galericulata ( migratory breeder ) , and 78.6% ( 121/154 ; 95% ci , 71.2~84.8% ) in anas crecca .
the prevalence of wild birds with an hi antibody titer above 1 : 20 or 1 : 1,280 were 86.7% and 21.2% , respectively .
wild birds captured in 2009 had a higher jev seropositive rate than those captured in 2007 ( fig .
2 ) . among the wild birds captured in 2009 that had an hi antibody titer of 1 : 20 or more , the most frequent hi titer was 1 : 640 ( 16.2% ) .
climate change , global warming , environmental destruction , and rapid urbanization can alter wild bird migratory paths and increase the incidence of arboviral disease outbreaks . under these conditions ,
vector - borne diseases caused by jev , wnv , and tbev may be easily introduced into disease - free countries by migratory wild birds . since jev was first isolated in korea in 1946 ,
several korean jev isolates have been reported and characterized based on a c / prm and e gene nucleotide analysis .
phylogenetic analyses to determine the genetic relationships between jev isolates found that korean jev isolates changed from genotype 3 to genotype 1 around 1993 .
however , few studies have addressed the possibility of jev introduction into korea via wild birds .
thus , we conducted a nationwide survey of jev antibodies in wild birds , including migratory birds . in this study ,
the sera of wild birds that were captured in 16 locations of the korean peninsula were tested for the presence of jev antibodies .
this hi test result was surprisingly high and suggested that most of the wild birds we captured in korea had been exposed to jev .
saito et al .   reported that 85.9% and 5.4% of wild ducks captured in hokkaido , japan have antibodies against jev according to the focus - reduction neutralization test ( frnt50 and frnt90 ) , which is more specific than the hi test .
additionally , 7.2% , 20.4% , and 28.1% of wild birds in 2007 , 2008 , and 2009 , respectively , had a titer > 1 : 1,280 .
sugiura and shimada   reported that horses experimentally inoculated with jev had an antibody titer of 1 : 1,280 within 2 or 3 weeks after inoculation .
therefore , wild birds with titers of 1 : 1,280 or higher may have been infected with wild jev within 3 weeks . although we conducted our serosurveillance study with an hi test , which is known to be less specific than the frnt
, our survey results correspond well with those of a previous study in wild ducks in japan . however , cross - reaction between jev and wnv in serodiagnoses is a well - known occurrence in flavivirus - infected animals , including birds .
therefore , it is necessary to perform a more specific method , such as a plaque - reduction neutralization test ( prnt ) , for jev serosurveillance in birds .
several positive jev serosurveillance results have been reported in different animals including swine , goats , cattle , horses , and wild boars in japan and korea .
it has been suggested that goats may serve as good sentinel animals for serological monitoring of jev infection because they are currently not vaccinated against jev in korea . unlike domestic animals ,
wild birds can not be inoculated with a jev vaccine , but they are easily exposed to several kinds of arthropods such as mosquitoes , ticks , and midges . the high number of positive jev titers in this study does not indicate viral intrusion into korea since jev viremia ends when jev - specific antibodies are detected in ducks .
however , wild birds with viremia may carry jev to several countries . according to data obtained from the korean center for disease control and prevention , 45 jev cases in human
more than half of these cases occurred in 2010 , suggesting that climate change or a high jev infection rate among wild birds may , in part , explain the increasing jev infection rate in the human population .
gyeonggi - do had a high incidence rate ( 42.3% ) of jev infection in humans , but no significant relationship was found between the seropositive rates in the capture areas and the jev incidence rate in humans .
nevertheless , new jev genotypes may be introduced into korea via wild or migratory birds .
it is therefore necessary to monitor jev antibodies using the prnt in wild birds . taken together , these findings lead us to predict that jev infections may appear in domestic animals such as pigs . in conclusion , the results of the present study suggested that wild birds with a high incidence rate ( 86.7% ) of jev antibodies may be introducing new jev genotypes into korea .
further studies are needed to isolate all jev genotypes from blood obtained from migratory birds .
various research projects examining disease distribution and transmission associated with climate change in the korean peninsula should also be carried out in the near future .
 climate change induced by recent global warming may have a significant impact on vector - borne and zoonotic diseases . 
 for example , the distribution of japanese encephalitis virus ( jev ) has expanded into new regions . 
 we surveyed the levels of hemagglutination - inhibition ( hi ) antibodies against jev ( family flaviviridae , genus flavivirus ) in wild birds captured in korea . 
 blood samples were collected from 1,316 wild birds including the following migratory birds : oceanodroma castro ( n = 4 ) , anas formosa ( n = 7 ) , anas penelope ( n = 20 ) , fulica atra ( n = 30 ) , anas acuta ( n = 89 ) , anas crecca ( n = 154 ) , anas platyrhynchos ( n = 214 ) , aix galericulata ( n = 310 ) , and anas poecilorhyncha ( n = 488 ) . all were captured in 16 locations in several korea provinces between april 2007 and december 2009 . out of the 1,316 serum samples tested , 1,141 ( 86.7% ) were positive for jev . 
 wild birds captured in 2009 had a higher seroprevalence of ant - jev antibodies than those captured in 2007 . wild birds with an hi antibody titer of 1 : 1,280 or higher accounted for 21.2% ( 280/1,316 ) of the animals tested . 
 these findings indicated that wild birds from the region examined in our study have been exposed to jev and may pose a high risk for introducing a new jev genotype into korea . 
Introduction
Materials and Methods
Collection of serum samples from wild birds
Hemagglutination inhibition (HI) test
Statistical analysis
Results
Discussion