BACKGROUND
the ebola virus  is an enveloped, negative-strand rna virus belonging to the family filoviridae in the order of mononegavirale <cit> .
four of the five ebolavirus species, zaire , sudan, tai forest, and the recently discovered bundibugyo ebolavirus, are endemic in continental africa and cause a severe form of viral hemorrhagic fever with high mortality in humans and non-human primates  <cit> .
reston ebolavirus  is sporadic in the philippines and has caused several epizootics in cynomolgus macaques  <cit> .
rebov was first isolated in  <dig> from cynomolgus macaques imported from the philippines for medical research in the united states  <cit> .
about  <dig>  monkeys died or were euthanized in a quarantine facility in reston, virginia.
subsequently,  <dig> animal handlers at the philippine exporter and four employees of the quarantine facility were found to have antibodies to the virus, indicating that they had been infected  <cit> .
epizootics in monkeys in the philippines were then reported in  <dig> and  <dig>  and all the epizootics have been traced back to a single monkey facility, in calamba, laguna in the philippines  <cit> .
since the closure of the facility in  <dig>  no rebov epizootics in cynomolgus monkeys have been reported.
in october  <dig>  rebov infection was confirmed for the first time in swine associated with multiple epizootics of respiratory and abortion-related diseases in the philippines  <cit> .
in several pools of swine samples collected from geographically distant swine farms, co-infection with rebov and porcine reproductive and respiratory syndrome virus  was confirmed  <cit> .
serological studies of limited scale on  <dig> swine sera in the affected farms failed to detect rebov antibodies in elisa, although prrsv antibodies were detected  <cit> .
it is still unclear how rebov was spread among swine during the epizootic.
moreover, it is not clear if rebov infection in the swine population is either sporadic and incidental or common in the philippines.
to try to answer these questions, we prepared multiple serodiagnosis systems for detecting rebov infection in swine and analyzed swine sera obtained from the affected farms and from farms not associated with any epizootics in the philippines.
the results showed a high prevalence of rebov infection in swine in the affected farms at the epizootics in 2008; however, rebov antibodies were not detected in the swine population not associated with the epizootics, indicating that rebov infection in swine in the philippines is not common, at least in some parts of tarlac.
RESULTS
detection of rebov-np and -gp antibodies in swine using ifa
swine sera were analyzed for the presence of rebov-np and -gp antibodies in ifa specific to rebov-np and -gp, respectively.
in the ifa, none of the  <dig> swine sera collected in japan showed a positive reaction , and so they were considered to be rebov-np and -gp antibody negative.
in the ifa specific to rebov-np, antibody positive swine sera showed characteristic granular staining patterns in the cytoplasm , which were indistinguishable from those of rebov-infected cynomolgus monkey sera  <cit>  and rebov-np immunized rabbit sera .
antibody-negative swine sera showed no reaction .
in the ifa specific to rebov-gp, antibody positive swine sera showed characteristic cellular surface staining patterns , which were indistinguishable from those of rebov-infected cynomolgus monkey sera and rebov-gp immunized rabbit sera .
antibody-negative swine sera showed no reaction .
in bulacan,  <dig>  and  <dig>  of the  <dig> swine sera showed positive reactions in the np- and gp-specific ifa, respectively.
in pangasinan, each  <dig>  of the  <dig> swine sera showed positive reactions in the np- and gp-specific ifa.
in total,  <dig>  and  <dig>  of the  <dig> swine sera collected at the affected farms were rebov-np and -gp antibody positive in the ifa, respectively .
on the other hand, none of the  <dig> swine sera collected in tarlac in the philippines showed any positive reaction .
detection of rebov neutralizing antibodies in swine
the vsv-pseudotype bearing rebov-gp efficiently infected vero e <dig> cells which are known to be susceptible to rebov infection.
the infectious titer of the vsv-pseudotype reached  <dig>  x 106 iu/ml when measured on vero e <dig> cells.
infection with the vsv-pseudotype was neutralized with rabbit serum to rebov-gp .
swine sera were analyzed in the nt using the vsv-pseudotype.
in the nt, none of the  <dig> swine sera collected in japan neutralized the infection with the vsv-pseudotype on vero e <dig> cells at serum dilutions of  <dig> in  <dig>  these were therefore considered to be rebov nt antibody negative.
in the affected farms,  <dig>  of the  <dig> swine sera in bulacan and  <dig>  of the  <dig> swine sera in pangasinan showed positive reactions in the nt, respectively at a serum dilution of  <dig> in  <dig>  in total,  <dig>  of the  <dig> swine sera in the affected farms showed nt antibody positive .
nt titers of the sera were then obtained for the  <dig> nt antibody positive sera in the affected farms.
nt titers ranged between  <dig> and  <dig>  with average of  <dig> and median of  <dig> .
in tarlac, none of the  <dig> swine sera showed any positive reaction .
detection of rebov-np and -gp specific antibodies in swine using igg-elisa
igg antibodies to the rebov-np and -gp were detected by recombinant rebov protein-based igg-elisa.
the od values to the np and gp of each serum sample at a dilution of  <dig> in  <dig> were plotted , and roc and tg-roc curves were also drawn .
since the od values of negative samples were very low, the cut off values defined as the values at intersection points of the sensitivity curve and specificity curve were  <dig>  and  <dig>  for rebov-np and -gp, respectively.
at the cut off values, sensitivity and specificity of the assay are over 95% in both igg-elisas .
in bulacan,  <dig>  and  <dig>  of the  <dig> swine sera showed positive reactions in the np- and gp-specific igg-elisa, respectively.
in pangasinan,  <dig>  and  <dig>  of the  <dig> swine sera showed positive reactions in the np- and gp-specific igg-elisa, respectively.
in total,  <dig>  and  <dig>  of the  <dig> swine sera collected at the affected farms were rebov-np and -gp antibody positive in the igg-elisa, respectively .
conversely,  <dig> of the  <dig> swine sera collected in tarlac in the philippines showed a positive reaction in the gp specific igg-elisa and  <dig> of the  <dig> swine sera collected in japan showed a positive reaction in the np- and gp-specific igg-elisa .
however, these results are considered to be false positives because the od values were close to the cut off level defined by the assay, additionally all the samples collected in tarlac and japan tested using the alternative serological assays were negative.
agreement between the serological assays detecting rebov antibodies
we next examined the agreement between the serological assays used in the present study.
the data obtained in each assay were binarized to either positive or negative result.
these binarized data were then statistically analyzed by pair-wise comparisons in nonparametric one-way anova.
this analysis showed that there were no significant differences between the serological assays .
the result supports the contention that all of the five serological assays used in the present study posses similar discriminatory capacity.
thus we are confident that many rebov antibody positive samples showed a positive reaction in all of the five assays.
discussion
zebov and marburg virus were identified in african fruit bat species  <cit> .
more recently, marburg virus was successfully isolated from a fruit bat, rousettus aegyptiacus <cit> , suggesting that fruit bats are reservoir animals of filoviruses.
in the philippines, we have recently demonstrated that an asian fruit bat, rousettus amplexicaudatus, has antibodies to rebov  <cit> .
since the rebov genome has not yet been detected in the bat, conclusive evidence that the bat species is a reservoir, or one of the reservoir animals, of rebov is not available.
nevertheless, it is possible that rebov was transmitted to swine from these bats since these bats inhabit many areas of the country, including the regions around the affected facilities both in pangasinan and bulacan.
in this study, we have aimed to clarify how rebov infection was spread among swine during the rebov epizootics.
we have tested  <dig> swine sera collected from the rebov affected farms using ifa and igg-elisa specific for rebov-np and -gp, and nt.
nt is the gold standard of serological assay in many virus infections.
however, since rebov needs to be cultured in high-containment laboratories, we performed an alternative nt using the vsv-pseudotype bearing rebov-gp.
this avoided the use of infectious rebov, enabling the work to be carried out at low containment.
previously it has been shown that vsv-pseudotype bearing ebolavirus gp mimicks ebolavirus infection  <cit> , approxymately 70% of the swine sera from rebov affected farms were rebov antibody positive.
this indicated that swine are susceptible to rebov infection.
unfortunately, we could not analyze the igm antibody responses in swine, since after the sera were heat inactivated the gamma globulin fractions were precipitated with ammonium sulfate, and reconstituted in pbs prior to be testing.
an indication of the igm responses to rebov would have provided evidence of a recent infection.
thus, it is still unclear if rebov infection was spread during epizootics or whether a population of the animals in the farms was infected with rebov prior to the epizootics.
the swine not associated with the epizootics, in tarlac, are considered to be free from rebov infection.
these samples were collected in  <dig>  over 2 years after the epizootic, and from animals born after the epizootic.
moreover, we could not analyze the swine specimens near the affected farms in  <dig>  thus, in this study, it is not clear if rebov infection in  <dig> was limited in the affected farms.
further study is necessary to conclude if the swine population in the philippines is generally free from rebov infection.
recently, it has been shown that the experimental infection of swine with rebov alone resulted in subclinical infection with rapid clearance of the virus  <cit> .
alternatively, zebov has been shown to replicate to high titers in experimentally infected swine and to cause severe lung pathology resulting in transmission of the virus to naïve animals  <cit> .
thus, swine has been shown experimentally to be highly susceptible to zebov infection.
furthermore, some amino acid mutations in np and/or vp <dig> in zebov resulted in adaptation of the virus to guinea pigs and mice  <cit> .
thus, we cannot rule out the possibility that mutations introduced in the rebov genome during serial transmission in swine will result in adaptation of the virus to swine in future.
in this regard, a regular serological survey of rebov infection in swine in the philippines is desirable.
the serodiagnosis systems presented in this study might be useful for such a survey.
CONCLUSIONS
the high prevalence of rebov infection in swine at the affected farms in  <dig> suggests that swine are susceptible for rebov infection.
the multiple serological assays used in the study are thought to be useful for future surveillance of reobv infection in swine in the philippines.

 the ebola virus is an enveloped, negative-strand rna virus belonging to the family filoviridae in the order of mononegavirale. four of the five ebolavirus species, zaire, sudan, tai forest, and the recently discovered bundibugyo ebolavirus, are endemic in continental africa and cause a severe form of viral hemorrhagic fever with high mortality in humans and non-human primates. reston ebolavirus is sporadic in the philippines and has caused several epizootics in cynomolgus macaques. rebov was first isolated in from cynomolgus macaques imported from the philippines for medical research in the united states. about monkeys died or were euthanized in a quarantine facility in reston, virginia. subsequently, animal handlers at the philippine exporter and four employees of the quarantine facility were found to have antibodies to the virus, indicating that they had been infected. epizootics in monkeys in the philippines were then reported in and and all the epizootics have been traced back to a single monkey facility, in calamba, laguna in the philippines. since the closure of the facility in no rebov epizootics in cynomolgus monkeys have been reported. in october rebov infection was confirmed for the first time in swine associated with multiple epizootics of respiratory and abortion-related diseases in the philippines. in several pools of swine samples collected from geographically distant swine farms, co-infection with rebov and porcine reproductive and respiratory syndrome virus was confirmed. serological studies of limited scale on swine sera in the affected farms failed to detect rebov antibodies in elisa, although prrsv antibodies were detected. it is still unclear how rebov was spread among swine during the epizootic. moreover, it is not clear if rebov infection in the swine population is either sporadic and incidental or common in the philippines. to try to answer these questions, we prepared multiple serodiagnosis systems for detecting rebov infection in swine and analyzed swine sera obtained from the affected farms and from farms not associated with any epizootics in the philippines. the results showed a high prevalence of rebov infection in swine in the affected farms at the epizootics in 2008; however, rebov antibodies were not detected in the swine population not associated with the epizootics, indicating that rebov infection in swine in the philippines is not common, at least in some parts of tarlac.  detection of rebov-np and -gp antibodies in swine using ifa swine sera were analyzed for the presence of rebov-np and -gp antibodies in ifa specific to rebov-np and -gp, respectively. in the ifa, none of the swine sera collected in japan showed a positive reaction, and so they were considered to be rebov-np and -gp antibody negative. in the ifa specific to rebov-np, antibody positive swine sera showed characteristic granular staining patterns in the cytoplasm, which were indistinguishable from those of rebov-infected cynomolgus monkey sera and rebov-np immunized rabbit sera. antibody-negative swine sera showed no reaction. in the ifa specific to rebov-gp, antibody positive swine sera showed characteristic cellular surface staining patterns, which were indistinguishable from those of rebov-infected cynomolgus monkey sera and rebov-gp immunized rabbit sera. antibody-negative swine sera showed no reaction. in bulacan, and of the swine sera showed positive reactions in the np- and gp-specific ifa, respectively. in pangasinan, each of the swine sera showed positive reactions in the np- and gp-specific ifa. in total, and of the swine sera collected at the affected farms were rebov-np and -gp antibody positive in the ifa, respectively. on the other hand, none of the swine sera collected in tarlac in the philippines showed any positive reaction. detection of rebov neutralizing antibodies in swine the vsv-pseudotype bearing rebov-gp efficiently infected vero e cells which are known to be susceptible to rebov infection. the infectious titer of the vsv-pseudotype reached x 106 iu/ml when measured on vero e cells. infection with the vsv-pseudotype was neutralized with rabbit serum to rebov-gp. swine sera were analyzed in the nt using the vsv-pseudotype. in the nt, none of the swine sera collected in japan neutralized the infection with the vsv-pseudotype on vero e cells at serum dilutions of in these were therefore considered to be rebov nt antibody negative. in the affected farms, of the swine sera in bulacan and of the swine sera in pangasinan showed positive reactions in the nt, respectively at a serum dilution of in in total, of the swine sera in the affected farms showed nt antibody positive. nt titers of the sera were then obtained for the nt antibody positive sera in the affected farms. nt titers ranged between and with average of and median of. in tarlac, none of the swine sera showed any positive reaction. detection of rebov-np and -gp specific antibodies in swine using igg-elisa igg antibodies to the rebov-np and -gp were detected by recombinant rebov protein-based igg-elisa. the od values to the np and gp of each serum sample at a dilution of in were plotted, and roc and tg-roc curves were also drawn. since the od values of negative samples were very low, the cut off values defined as the values at intersection points of the sensitivity curve and specificity curve were and for rebov-np and -gp, respectively. at the cut off values, sensitivity and specificity of the assay are over 95% in both igg-elisas. in bulacan, and of the swine sera showed positive reactions in the np- and gp-specific igg-elisa, respectively. in pangasinan, and of the swine sera showed positive reactions in the np- and gp-specific igg-elisa, respectively. in total, and of the swine sera collected at the affected farms were rebov-np and -gp antibody positive in the igg-elisa, respectively. conversely, of the swine sera collected in tarlac in the philippines showed a positive reaction in the gp specific igg-elisa and of the swine sera collected in japan showed a positive reaction in the np- and gp-specific igg-elisa. however, these results are considered to be false positives because the od values were close to the cut off level defined by the assay, additionally all the samples collected in tarlac and japan tested using the alternative serological assays were negative. agreement between the serological assays detecting rebov antibodies we next examined the agreement between the serological assays used in the present study. the data obtained in each assay were binarized to either positive or negative result. these binarized data were then statistically analyzed by pair-wise comparisons in nonparametric one-way anova. this analysis showed that there were no significant differences between the serological assays. the result supports the contention that all of the five serological assays used in the present study posses similar discriminatory capacity. thus we are confident that many rebov antibody positive samples showed a positive reaction in all of the five assays. discussion zebov and marburg virus were identified in african fruit bat species. more recently, marburg virus was successfully isolated from a fruit bat, rousettus aegyptiacus, suggesting that fruit bats are reservoir animals of filoviruses. in the philippines, we have recently demonstrated that an asian fruit bat, rousettus amplexicaudatus, has antibodies to rebov. since the rebov genome has not yet been detected in the bat, conclusive evidence that the bat species is a reservoir, or one of the reservoir animals, of rebov is not available. nevertheless, it is possible that rebov was transmitted to swine from these bats since these bats inhabit many areas of the country, including the regions around the affected facilities both in pangasinan and bulacan. in this study, we have aimed to clarify how rebov infection was spread among swine during the rebov epizootics. we have tested swine sera collected from the rebov affected farms using ifa and igg-elisa specific for rebov-np and -gp, and nt. nt is the gold standard of serological assay in many virus infections. however, since rebov needs to be cultured in high-containment laboratories, we performed an alternative nt using the vsv-pseudotype bearing rebov-gp. this avoided the use of infectious rebov, enabling the work to be carried out at low containment. previously it has been shown that vsv-pseudotype bearing ebolavirus gp mimicks ebolavirus infection, approxymately 70% of the swine sera from rebov affected farms were rebov antibody positive. this indicated that swine are susceptible to rebov infection. unfortunately, we could not analyze the igm antibody responses in swine, since after the sera were heat inactivated the gamma globulin fractions were precipitated with ammonium sulfate, and reconstituted in pbs prior to be testing. an indication of the igm responses to rebov would have provided evidence of a recent infection. thus, it is still unclear if rebov infection was spread during epizootics or whether a population of the animals in the farms was infected with rebov prior to the epizootics. the swine not associated with the epizootics, in tarlac, are considered to be free from rebov infection. these samples were collected in over 2 years after the epizootic, and from animals born after the epizootic. moreover, we could not analyze the swine specimens near the affected farms in thus, in this study, it is not clear if rebov infection in was limited in the affected farms. further study is necessary to conclude if the swine population in the philippines is generally free from rebov infection. recently, it has been shown that the experimental infection of swine with rebov alone resulted in subclinical infection with rapid clearance of the virus. alternatively, zebov has been shown to replicate to high titers in experimentally infected swine and to cause severe lung pathology resulting in transmission of the virus to naïve animals. thus, swine has been shown experimentally to be highly susceptible to zebov infection. furthermore, some amino acid mutations in np and/or vp in zebov resulted in adaptation of the virus to guinea pigs and mice. thus, we cannot rule out the possibility that mutations introduced in the rebov genome during serial transmission in swine will result in adaptation of the virus to swine in future. in this regard, a regular serological survey of rebov infection in swine in the philippines is desirable. the serodiagnosis systems presented in this study might be useful for such a survey.  the high prevalence of rebov infection in swine at the affected farms in suggests that swine are susceptible for rebov infection. the multiple serological assays used in the study are thought to be useful for future surveillance of reobv infection in swine in the philippines. 
reston ebolavirus has caused several epizootics in cynomolgus monkeys but is not associated with any human disease. the high prevalence of rebov infection in swine in the affected farms in suggests that swine is susceptible for rebov infection. a total of swine sera collected at two rebov-affected farms in in pangasinan and bulacan, were tested for the presence of rebov-specific antibodies using multiple serodiagnosis systems. a total of swine sera collected in a non-epizootic region, tarlac, were also tested to clarify the prevalence of rebov infection in the general swine population in the philippines. some 70 % of swine sera at the affected farms were positive for rebov antibodies in the multiple serodiagnosis systems. on the other hand, none of the swine sera collected in tarlac showed positive reactions in any of the diagnosis systems. the multiple serological assays used in the study are thought to be useful for future surveillance of reobv infection in swine in the philippines. in late rebov infections were identified in swine for the first time in the philippines. ebola viruses cause viral hemorrhagic fever in humans and non-human primates and are endemic in africa.
 ebola viruses cause viral hemorrhagic fever in humans and non-human primates and are endemic in africa. reston ebolavirus has caused several epizootics in cynomolgus monkeys but is not associated with any human disease. in late rebov infections were identified in swine for the first time in the philippines. methods a total of swine sera collected at two rebov-affected farms in in pangasinan and bulacan, were tested for the presence of rebov-specific antibodies using multiple serodiagnosis systems. a total of swine sera collected in a non-epizootic region, tarlac, were also tested to clarify the prevalence of rebov infection in the general swine population in the philippines.  some 70 % of swine sera at the affected farms were positive for rebov antibodies in the multiple serodiagnosis systems. on the other hand, none of the swine sera collected in tarlac showed positive reactions in any of the diagnosis systems.  the high prevalence of rebov infection in swine in the affected farms in suggests that swine is susceptible for rebov infection. the multiple serological assays used in the study are thought to be useful for future surveillance of reobv infection in swine in the philippines. reston ebolavirusantibodyswinephilippines 