most commonly , hantaviruses are transmitted to humans via aerosols of infectious excreta ( urine , feces , saliva ) from chronically infected wild rodents ( 6 ) .
the old world hantaviruses are most often associated with hemorrhagic fever with renal syndrome ( hfrs ) , while the new world hantaviruses cause hantavirus pulmonary syndrome ( hps ) .
this dichotomy , however , is not completely clear in all cases ( 7 ) .
the natural reservoirs of hantaviruses are small rodents or insectivores , and the virus is believed to cause asymptomatic life - long infection in these animals .
hantaviruses are usually species - specific , although spill - overs have been described ( 8) . puumala hantavirus ( puuv ) causes the majority of hantavirus - related clinical cases in europe .
puuv causes a mild form of hfrs and is carried and shed by the bank vole ( myodes glareolus ) ( 9 ) .
other hfrs - causing hantaviruses circulating in europe include dobrava and saaremaa hantaviruses , which are carried by the yellow - necked mouse ( apodemus flavicollis ) and the striped field mouse ( a. agrarius ) , respectively ( 6 ) .
seov is the only known hantavirus with a potentially world - wide distribution due to the ubiquitous occurrence of its reservoir hosts , the brown and the black rat ( rattus norvegicus and r. rattus , respectively ) ( 10 ) .
nevertheless , prior to the last decade , human and wild rat infections of seov were , for the most part , detected only in asia with human infections in europe limited to laboratory rat handling ( 4 ) . during recent years , however , seov has been detected in wild rats in france , belgium , and the united kingdom ( 2 , 3 ) , and in pet rats in sweden , england , and wales ( 1 , 4 ) . human cases of severe seov - caused hfrs have been reported in france and the united kingdom ( 3 , 5 ) . in neither of these severe seov cases were there any connections to pet or laboratory rats , strongly indicating that the infections were contracted from nature .
this study proves the occurrence of seov in the wild rat population in the netherlands .
analyses were conducted at the zoonosis science center at the department of medical biochemistry and microbiology , uppsala university , the public health agency of sweden , stockholm , sweden , and at the department of microbiology , graduate school of medicine , hokkaido university , japan .
rat hearts were vortexed together with 1 ml of phosphate - buffered saline ( pbs ) and centrifuged .
the samples were initially screened by 1 ) enzyme - linked immunosorbent assay ( elisa ) , 2 ) indirect immunofluorescence assay ( ifa ) , and 3 ) immunoblotting ( ib):an in - house seov elisa ( will be described elsewhere , verner - carlsson , unpublished ) was used for initial screening .
briefly , elisa - plates were coated by the hantavirus - reactive bank vole mab 1c12 ( 4 g / ml ) , followed by post - coating ( 3% bovine serum albumin ( bsa ) in pbs ) , and incubation of seov ( strain 8039 ) native antigen .
rat samples were diluted 1:16 ( equivalent to a final serum dilution of 1:400 ) and rat control sera were incubated at a dilution of 1:400 , followed by alkaline phosphatase ( alp)-conjugated goat anti - rat antibodies ( jackson immunoresearch ) .
all incubations lasted 1 h at 37c , and the plates were washed four times between each step .
cut - off was set to od 0.100 at 405 nm.ifa using seov ( strain sr-11 ) infected vero e6 cells was used as described earlier ( 11 ) .
briefly , rat blood collected on filter paper was diluted in pbs and used for ifa analyses and ib analyses .
serum from a wistar rat experimentally infected with seov ( strain sr-11 ) was used as positive control .
rat samples were diluted 1:50 , and control rat serum was diluted 1:100 and put onto glass slides with prefixed seov - infected vero e6 cells for 1 h at room temperature .
after washing , fitc - conjugated goat anti - rat antibodies were incubated for 1 h at room temperature .
fluorescence staining was visualized by an immunofluorescence microscope.the samples were further subjected to ib using seov ( strain sr-11 ) infected vero e6 cells and uninfected vero e6 cells as antigens , as described earlier ( 12 ) .
briefly , after transfer onto pvdf membranes , the membranes were blocked in block ace buffer ( ds pharma biomedical ) for 1 h at rt .
the membranes were subsequently incubated with the rat samples diluted 1:50 and positive / negative control rat serum at 1:100 dilutions , for 1 h rt , using a microwell system , followed by the addition of horseradish peroxidase - conjugated goat anti - rat igg for 1 h at rt .
an in - house seov elisa ( will be described elsewhere , verner - carlsson , unpublished ) was used for initial screening .
briefly , elisa - plates were coated by the hantavirus - reactive bank vole mab 1c12 ( 4 g / ml ) , followed by post - coating ( 3% bovine serum albumin ( bsa ) in pbs ) , and incubation of seov ( strain 8039 ) native antigen .
rat samples were diluted 1:16 ( equivalent to a final serum dilution of 1:400 ) and rat control sera were incubated at a dilution of 1:400 , followed by alkaline phosphatase ( alp)-conjugated goat anti - rat antibodies ( jackson immunoresearch ) .
all incubations lasted 1 h at 37c , and the plates were washed four times between each step . cut - off was set to od 0.100 at 405 nm .
ifa using seov ( strain sr-11 ) infected vero e6 cells was used as described earlier ( 11 ) .
briefly , rat blood collected on filter paper was diluted in pbs and used for ifa analyses and ib analyses .
serum from a wistar rat experimentally infected with seov ( strain sr-11 ) was used as positive control .
rat samples were diluted 1:50 , and control rat serum was diluted 1:100 and put onto glass slides with prefixed seov - infected vero e6 cells for 1 h at room temperature .
after washing , fitc - conjugated goat anti - rat antibodies were incubated for 1 h at room temperature .
the samples were further subjected to ib using seov ( strain sr-11 ) infected vero e6 cells and uninfected vero e6 cells as antigens , as described earlier ( 12 ) .
briefly , after transfer onto pvdf membranes , the membranes were blocked in block ace buffer ( ds pharma biomedical ) for 1 h at rt .
the membranes were subsequently incubated with the rat samples diluted 1:50 and positive / negative control rat serum at 1:100 dilutions , for 1 h rt , using a microwell system , followed by the addition of horseradish peroxidase - conjugated goat anti - rat igg for 1 h at rt .
two out of 16 samples , # 22 and # 33 , were found clearly positive by all three screening methods ; rat # 84 was positive by elisa and ifa , but negative in ib ( table 1 ) . to finally confirm the hantavirus reactivity and a seov - specificity , we tested the three positive samples # 22 , # 33 , and # 84 by frnt , as previously described ( 13 ) .
briefly , the rat samples were serially diluted and mixed with diluted viruses containing 3070 focus forming units/100 l .
confluent vero e6 cell monolayers in six - well tissue culture plates were used and incubated at 37c for 1 h after addition of the virus / antibody solution . to create a secluded environment ,
a mixture of tissue culture and agarose medium was subsequently added to the wells , which were then incubated for 8 days . to ensure visibility of the results ,
the agarose was removed from the wells , and the cells were fixed with methanol . to indicate virus - infected cells , the anti - hantavirus bank vole mab 1c12 followed by peroxidase - labeled goat antibodies to mouse igg ( jackson )
3 , 3 , 5 , 5-tetramethylbenzidine substrate ( sigma ) was used as substrate , and foci were enumerated .
the samples # 22 , # 33 and # 84 all showed high titers of neutralizing antibodies ( table 1 ) .
summary of elisa , ifa , ib , frnt , and pcr results 
  elisa : enzyme - linked immunosorbent assay ; ifa : immunofluorescence assay ; ib : immunoblotting ; frnt : focus reduction neutralization test ; pcr : polymerase chain reaction ; nt : not tested ; od : optical density at 405 nm , adjusted to the positive control=1.00 .
a hantavirus genus - specific nested rt - pcr was applied as described earlier ( 14 ) .
samples # 22 , # 33 and # 84 all showed positive results , while the serologically negative sample # 31 was found negative ( table 1 ) .
this is the first discovery of seov - specific antibodies in wild rats in the netherlands . in previous attempts to discover seov , during 20082011 ,
161 brown rats and 61 black rats were trapped at different types of environments ( farmlands , urban locations , and nature reserves ) in rijn and ijssel regions in the netherlands . despite initial findings of seov - reactive antibodies in several animals based on an adapted commercial ifa , and two different elisas , seov - infection could never be confirmed by neutralization test or viral sequence data ( data not shown ) .
it is therefore possible that the dispersal of seov in the area has happened relatively recently .
whereas the detection of antibodies can , in case of a number of pathogens , indicate a previous infection that is no longer an infectious threat , similar conclusions can not be made concerning hantaviruses . as demonstrated in the case of puuv infections , hantaviruses can be secreted by bank voles for several months after the initial infection ( 15 ) , lundkvist et al . , unpublished observations ) .
furthermore , even at room temperatures , the virus is stable and infectious over a few weeks outside the rodent ( 16 ) making virus transmission control a huge challenge .
as seen from the example of west nile virus emergence in the united states , the existence of wildlife reservoirs makes eradication of a disease that has had time to establish itself unrealistic .
thus , the recommendations for handling the situation must be made with a long - term perspective . since aerosols , contaminated by virus - infected rodent excreta ,
are the main source for human infection with hantaviruses , actual physical contact between rodent and man is not necessary for disease transmission ( 6 ) .
consequently , in many cases it will be difficult for both patients and medical specialists to suspect transmission of seov .
therefore , disseminating information about the occurrence of seov in wild rat populations in the netherlands has great importance for both medical workers and the general public .
in addition , tailored messages are needed for people exposed to seov due to their working environments . transmission 
preventive measures in the form of gloves and protective facemasks may be necessary for people working in rat - infested environments , particularly for professional rat controllers .
steps have been taken by the netherlands centre for infectious disease control ( rivm ) to provide this type of information .
further studies are planned to collect evidence on the circulation of this hantavirus among the wild rat population in other geographical areas in the netherlands , to generate and compare dutch seov sequences to seov from other countries , while the occurrence of human infections will be assessed through increased surveillance of clinical cases with hfrs - like symptoms and sero - surveillance of , for example , blood donors .
the recent discoveries of seov circulating in wild rat populations in france , belgium , and the united kingdom ( 2 , 3 ) , and now also in the netherlands , together with the cases of severe human seov - infections so far confirmed in the united kingdom and france ( 1 , 4 ) , strongly highlight the importance of further studies all over europe .
the authors have not received any funding or benefits from industry or elsewhere to conduct this study .
 we report the first detection of seoul hantavirus ( seov)-specific antibodies in the wild brown rat population in the netherlands . 
 seov - reactive antibodies were found in three rats out of 16 in a repeated series of tests including immunofluorescence assay , immunoblot , and enzyme - linked immunosorbent assay . 
 focus reduction neutralization test confirmed the presence of seov - specific antibodies , and reverse - transcription polymerase chain reaction ( rt - pcr ) confirmed the presence of hantaviral rna . 
 this discovery follows the recent findings of seov infections in wild and pet brown rats and humans in england , wales , france , belgium , and sweden , indicating an even higher importance of this hantavirus for public health in large areas of europe . 
Background
Virus investigation
Discussion
Conflict of interest and funding