to estimate the risk for infections with mosquitoborne viruses , we screened the human population of the flooded area ( figure 1 ) for antibodies against the viruses known to occur in central europe ( 2 ) : tahyna ( tahv ) , orthobunyavirus of the california group , bunyaviridae ; west nile ( wnv ) , flavivirus of the japanese encephalitis group , flaviviridae ; sindbis ( sinv ) , alphavirus , togaviridae ) ; and batai ( batv ) , orthobunyavirus of the bunyamwera group , bunyaviridae .
floodplain forests identified on the landsat mss satellite images ( dotted red line ) , with hydrology and settlement in background ( dmu-200 , vtop dobruka ) , and proportion of tahya virus seropositive residents at particular localities ( large , medium , and small circles indicate the risk zones a , b , and c , respectively ) .
b ) [ inset ] , radar satellite image of the conflux of the labe and vltava rivers on august 17 , 2002 ( 2 days after the flood culmination ) , showing extent of floodwater ( dark areas ) .
inundated forests , with subsequent mass occurrences of ochlerotatus and aedes mosquitoes , are visible as lighter areas surrounding labe river upstream of the confluence ; scattered lagoons ( dark areas ) in arable fields along both rivers far left and right turned into breeding sites of predominantly culex mosquitoes .
zone a was a forested floodplain along the labe river between obstv - kly and lobkovice - kozly ( 11 villages ) , with large quantities of mosquitoes .
zone b was an intermediate area between zones a and c ( 5 villages , 1 small town ) , with fewer breeding sites but possibility for mosquito migration from zone a. zone c was the area along the vltava and labe rivers between kralupy and horn poaply ( 25 villages and small towns ) , with no floodplain forests and few breeding sites for mosquitoes .
zone d was a control zone , with only sporadic occurrences of mosquitoes ( mainly in prague ) .
informed written consent and serum samples were obtained from 497 survey participants of various ages from september 6 to september 13 , 2002 ( 3 weeks after the flood culmination and 2 weeks after the mosquito emergence ) .
paired serum samples were taken from 150 of the survey participants 6 months later , from april 9 to may 15 , 2003 ( 34 in zone a , 43 in zone b , 73 in zone c ) .
serologic examination was performed with the hemagglutination - inhibition ( hit ) and plaque - reduction neutralization tests ( prnt ) in microplates ( 35 ) .
the strains used for hit were tahv 92 , wnv eg101 , batv 184 , and sinv eg339 ; a commercial control antigen ( test - line ltd .
, brno , czech republic ) of central european tickborne encephalitis virus ( ceev ) was used .
all serum samples were acetone - extracted and tested with sucrose- and acetone - processed antigens by using 8 hemagglutinin units ; titers > 20 were considered positive . for prnt , tahv t16 , wnv eg101 , ceev hypr , and batv , 184 viral strains were used .
the test was conducted on vero or spev ( embryonic pig kidney : for ceev ) cells .
all serum samples were heat inactivated and screened for antibodies at 1:8 ; those reducing the number of virus plaques by 90% were considered positive and titrated to estimate dilutions causing plaque  number reduction by 50% ( prnt50 ) and 90% ( prnt90 ) .
prnt with batv was used only as a confirmatory test for the serum samples reacting with batv in hit . against tahv , 82 ( 16.5% ) of 497 study participants
had neutralizing antibodies , and 74 ( 14.9% ) were seropositive in hit . in prnt50 ,
the titers were 322048 ( geometric mean titer [ gmt ] 260 ) , in prnt90 161024 ( gmt 119 ) , and in hit 2040 to 160 ( gmt 40 ) .
no difference occurred in neutralizing antibody prevalence between sexes , 32 ( 15.8% ) of 202 males and 50 ( 16.9% ) of 295 females (  = 0.11 ; p = 0.744 ) .
the prevalence rate increased significantly with age ( table  1 :  = 39,809 ; p < 0.001 ) ; tahv antibodies were found infrequently in persons < 19 years of age . neutralizing antibody distribution , with respect to the residence location ( table 2 , figure 1 ) , showed the highest seroprevalence in zone a ( 28% ) , lower seroprevalences in zones b and c , and 5% in the control zone d (  = 14.57 ; p = 0.002 ) .
significant differences were found between zone d and all other zones , and between zones a and c (  = 7.243 ; p = 0.007 ) , but not between zones a and b or b and c ; hit yielded similar results .
the seroprevalence in relation to the proximity of study participants locations to the nearest floodplain forest within zones a , b , and c demonstrated decreasing seroprevalence with increasing proximity to the forest (  = 8.51 ; p = 0.003 ) ( table 2 ) .
distribution of 50% plaque - reduction neutralization titers of antibodies to tahyna virus ( y axis , number of seroreactors ) . *
cb , central bohemia ; sm , southern moravia ; n , number of residents examined . 
source ( 6 ) . * as related to the residence location : risk zones a to d ; and distance to floodplain forest ( fpf , within zones a , b , and c only ) . 
although serum samples from 34 ( 6.8% ) study participants reacted in hit with the wnv at titers 40 to 80 , all of them also reacted with ceev at titers similar or higher ( 160 ) .
ceev could have occurred in the area , and some study participants may have been vaccinated against tickborne encephalitis . in prnt90 ,
6 study participants ( 1.2% ) reacted with wnv but at low titers of 8 to 16 ; these serum samples also reacted in prnt with ceev ; thus , the results were considered to be crossreactions as well .
additionally , 42 ( 8.5% ) seroreactors against wnv appeared in the less stringent prnt50 , but all titers were low ( 832 ) and cross - reacted with ceev . against sinv , antibodies were tested with hit only and detected in specimens from 7 ( 1.4% ) study participants , with low titers of 20 to 40 . of the batv ,
specimens from 7 study participants reacted in hit at a low titer of 20 . by confirmatory testing of these serum samples in prnt , only 1 ( 0.2% ) showed specific antibodies to batv ; the titer was 64 in prnt50 and 32 in prnt90 .
after the flood the infection episode occurred in one 55-year - old woman from obstv ( zone a ) , as shown by the seroconversion in both hit ( < 20/40 ) and prnt50 ( < 8/512 ) .
three other study participants seroconverted in 1 test only : a 40-year - old man from chlumn , zone b ( hit 20/80 ; prnt 128/128 ) ; a 32-year - old man from chlumn ( hit < 20/2040 ; prnt 128/64 ) ; and an 80-year - old woman from obstv ( hit 20/80 ; prnt 64/32 ) .
local general practitioners did not corroborate consistent signs of a disease reported by these 4 study participants from october 2002 to april 2003 .
in general , clinical symptoms of tahv infection are milder in adults than in children ( 7 ) .
seroconversion against mosquitoborne viruses was not detected in any of the 73 study participants in zone c.
on the basis of this serosurvey , recent infections with wnv ( in contrast to south moravia after the 1997 flood ( 5,6 ) , sinv , and batv have not been found in central bohemia after the flood .
however , activity of another mosquitoborne virus , tahv , has been found in a natural focus along the labe river at neratovice .
this focus has so far gone unnoticed ( 8) . lower frequency of tahv antibodies has been detected along the lower reaches of the vltava river .
the prevalence of antibodies to tahv increased with risk - zone ranking ( from zone d to the highest risk zone a ) and with decreasing distance to floodplain forests , the primary breeding habitat of vector mosquitoes ( 911 ) . in disease - endemic areas , the proportion of residents with antibodies against california group viruses increase with age ( 6,12 ) .
a similar situation occurred in the central bohemian flooded area , where antibodies to tahv were detected in a low proportion of residents < 20 years of age .
at least 1 seroconversion among 150 residents ( attack rate 0.67% ) against tahv has been proven . with 100,000 inhabitants in the risk zones ( 1992 census ) ,
670 ( 95% confidence interval 203,719 ) persons could have been infected after the flood .
environmental factors , such as heavy rains followed by a flood , artificial inundation of floodplain forests , or rehabilitation of wetlands that support mosquito - vector populations , could give rise to preconditions for an increased incidence of mosquitoborne infectious diseases , even in temperate climates . under such circumstances
, the optimum strategy is an epidemiologic surveillance that includes monitoring , especially of infection rate of mosquito populations and incidence of mosquitoborne diseases in humans .
 specimens from residents ( n = 497 ) of an area affected by the 2002 flood were examined serologically for mosquitoborne viruses . 
 antibodies were detected against tahyna ( 16% ) , sindbis ( 1% ) , and batai ( 0.2% ) viruses , but not west nile virus . 
 an examination of paired serum samples showed 1 tahyna bunyavirus ( california group ) infection . 
The Study
Conclusions