BACKGROUND
the family filoviridae includes three genera, ebolavirus, marburgvirus, and cuevavirus.
the genus ebolavirus currently has five members: bundibugyo virus , ebola virus , reston virus , sudan virus , and taï forest virus
<cit> .
filoviruses induce lethal viral hemorrhagic fevers  in both humans and non-human primates, while restv infection in humans is probably subclinical, yet it also causes highly lethal vhf in macaques
<cit> .
restv epizootics among cynomolgus macaques emerged in  <dig>   <dig>   <dig>  and  <dig>  in all of these epizootics, the cynomolgus macaques originated in a single primate breeding facility in the philippines
<cit> .
although the natural reservoir of restv remains unknown, restv was isolated from pigs in the philippines, in addition to porcine reproductive and respiratory syndrome virus  and porcine circovirus type- <dig> in 2008
<cit> .
considering the social impact of ebolaviruses, it is important to understand the endemic and epizootic status of restv in the philippines.
in this study, we investigated the antibody responses of cynomolgus macaques that could be dead-end hosts for restv.
using serum specimens collected from cynomolgus macaques during a restv outbreak in the philippines in  <dig>  we attempted to elucidate the significance of neutralizing antibodies to restv in viral clearance.
we have previously established an enzyme-linked immunosorbent assay  and an indirect immunofluorescent antibody assay  specific for restv nucleoprotein
<cit> .
these assays are useful tools for investigating the signs of restv infection in cynomolgus macaques.
in human cases, antibody responses against ebolaviruses have been analyzed extensively: igg responses to np and other structural proteins  have been shown to correlate with asymptomatic and surviving cases, and neutralizing antibody responses targeting the ebolaviruses glycoprotein  appear to be the major indicator of protective immunity
<cit> .
on the other hand, proinflammatory cytokines/chemokines are known to play a major role in the pathogenesis of ebolaviruses infections in various species.
previous studies have shown an uncontrolled secretion of proinflammatory cytokines/chemokines to contribute to a fatal outcome in ebov-infected humans
<cit>  and cynomolgus macaques
<cit> .
strong proinflammatory cytokine/chemokine responses are also observed in convalescent or asymptomatic cases
<cit> .
in restv-infected cynomolgus macaques, high viremia has been shown to induce the secretion of proinflammatory cytokines/chemokines
<cit> .
however, there have so far only been a limited number of studies on the impact of proinflammatory cytokine/chemokine responses in the convalescent phase of restv infection.
in this study, we grouped the cynomolgus macaque samples based on the presence of restv np-antigen in sera and analyzed the antibody reactions and cytokine/chemokine inductions to evaluate the presence of neutralizing antibody to restv.
we found that the anti-gp <dig>  responses, but not the anti-np responses, were closely correlated with the neutralization antibody responses, as well as the clearance of viremia, in the sera of restv-infected cynomolgus macaques.
additionally, a high concentration of proinflammatory cytokines/chemokines was detected in the convalescent phase specimens.
these data suggest that both the anti-gp <dig>  responses and proinflammatory cytokines/chemokines play significant roles in the recovery from restv infection in cynomolgus macaques.
RESULTS
restv np-and gp <dig> -specific antibodies, neutralizing antibody responses, and the viral antigens in the cynomolgus macaque sera from the  <dig> restv epizootic
twenty-seven serum samples derived from cynomolgus macaques that were either found already dead or had been euthanized at the facility were available.
the presence of restv np antigens was evaluated by antigen-capture elisa
<cit>  or immunohistochemistry
<cit> , while that of anti-restv np igg was evaluated using igg elisa and ifa methods
<cit> .
restv np antigens were detected in the liver in #  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig>  while restv np was detected by antigen capture elisa in the sera of # <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig>  we therefore assumed that these cynomolgus macaques had suffered from the acute viremic phase of the disease.
seventeen of the  <dig> samples  were positive for anti-np igg in igg elisa, while these samples all reacted in ifa.
on the other hand, two samples  were only positive in ifa.
we considered the samples as anti-np igg-positive when either elisa or ifa showed positive reaction.
as a result, a total of  <dig> samples had anti-np igg.
cynomolgus macaques with anti-np igg consisted of np antigen-positive  and np antigen-negative groups .
in order to examine whether the sera contained anti-gp <dig>  antibodies, we employed a gp <dig> -specific elisa and ifa
<cit> .
restv gp <dig> Δtm prepared by a baculovirus expression system and restv gp <dig> -expressing hela cells were used as antigens for gp <dig> -specific elisa and ifa, respectively.
anti-restv gp <dig>  igg were detected in  <dig>  out of the  <dig> serum samples according to elisa , whereas the remaining  <dig> samples  showed negative reactions.
nine serum samples positive for gp <dig>  antibodies in the igg-elisa also showed positive reactions in the ifa, while one serum sample  was only positive in the ifa.
serum samples showing positive reactions in either the gp <dig> -specific igg-elisa or ifa were considered to be anti-gp <dig>  positive .
interestingly, the sera derived from cynomolgus macaques in the acute viremic phase did not contain any detectable anti-gp <dig>  igg, although they often contained anti-np igg.
*nd: not determined, **euthanized: monkeys were euthanized regardless of clinical manifestation.
#nr: not recorded, ag +: antigen positive, ag -: antigen negative, nt+: neutralization antibody positive, nt-: neutralization antibody negative.
the specimens with case ids written in italics were considered in acute phase.
the specimens with case ids written in bold were considered in convalescent phase.
we next attempted to detect the neutralization  antibody response in the sera of restv-infected cynomolgus macaques.
the vsv pseudotype restv gp <dig>   was used for the nt assay
<cit> .
twelve serum samples  neutralized the vsv-restv-gp <dig> /gfp infection, with nt titers ranging from  <dig> and  <dig>  .
the anti-gp <dig>  igg were not detectable in # <dig> and # <dig> by igg elisa, while those samples both had a neutralizing activity.
these two specimens showed a positive response for viral antigen in the ag-capture elisa and were thus considered to be collected in an early seroconversion phase.
all cynomolgus macaques at the facility were euthanatized regardless of clinical status and there was a possibility that some of the cynomolgus macaques had combined infection with simian hemorrhagic fever virus  in the animal facility
<cit> .
therefore, immune responses against restv did not always reflect the clinical manifestation.
for these reasons, we defined “convalescent” or “non-convalescent” based only upon serological findings.
it is noteworthy that, among the serum samples that were positive for viral antigen and negative for the nt antibody , all nine serum samples were negative for anti-gp <dig>  antibodies, whereas only two samples were negative for anti-np antibodies.
on the other hand, all of the ten serum samples that were negative for viral antigen and positive for the nt antibody  were positive for both anti-gp <dig>  and anti-np antibodies.
this finding indicates that the anti-gp <dig>  antibody may therefore increase in cynomolgus macaques in the convalescent phase, while anti-gp <dig>  antibody is rarely detectable in the acute viremic phase of infection.
multiplex assay for cytokines and chemokines in the cynomolgus macaque sera
ebola virus infection triggers the expression of several proinflammatory cytokines/chemokines
<cit> .
to examine whether the convalescence from the restv infection correlates with the circulating proinflammatory cytokines/chemokines, eleven restv-infected cynomolgus macaque serum samples were subjected to a multiplex cytokine analysis.
since we do not know when the infection occurred for each cynomolgus macaque, it is still unclear whether the sera represented an early or late stage of infection.
we used seven convalescent phase sera , and four acute viremic phase sera .
among the  <dig> serum samples, only these  <dig> serum samples were available for multiplex assay.
since the sera were heat-inactivated at 56°c for  <dig> min prior to being subjected to the multiplex analysis, some cytokines, such as gm-csf and il- <dig>  which were previously shown to be elevated in some restv infected cynomolgus macaques
<cit> , could not be measured in the assay.
we found that concentrations of several proinflammatory cytokines/chemokines  were significantly higher in convalescent than in acute phase sera .
this observation is similar to the previous studies showing elevated concentrations of proinflammatory cytokines/chemokines in the convalescent or asymptomatic human cases
<cit> .
in contrast, the concentrations of the five cytokines/chemokines  did not differ significantly between the two groups .
furthermore, the concentration of mcp- <dig>  one of the proinflammatory chemokines, was lower in the convalescent than in the non-convalescent sera .
these data indicated that ifn-γ, il- <dig>  il- <dig>  il-1ra or mip1α might therefore be involved with the host immune responses in the convalescent phase of restv infection.
discussion
we previously developed a restv np-specific igg-elisa and ifa that proved to be useful for the seroepidemiological studies of cynomolgus macaques during the restv epizootic in the philippines in 1996
<cit> .
the assays based on recombinant np are sensitive for the detection of restv-specific antibodies.
on the other hand, anti-gp <dig>  antibodies are elicited in ebov-infected human cases and are believed to have protective roles against lethal ebov infection
<cit> .
in the present study, in order to gain insight into the igg responses during the recovery from infection with restv, anti-np, anti-gp <dig> , and neutralizing antibodies and the level of viremia in the serum specimens were analyzed.
the data presented herein showed that the anti-gp <dig>  response, rather than the anti-np response, was correlated with both the lack in viremia and the neutralizing activities in the sera of restv-infected cynomolgus macaques.
there may be at least two possibilities for the lack of anti-gp <dig>  igg in the acute phase samples.
it is known that soluble gp , which does not contain membrane anchor, is secreted during ebolaviruses infection, and it can absorb the anti-gp <dig>  antibodies
<cit> .
the other possibility is that apoptosis of lymphocytes is induced during restv infection and the resulting host immune responses may thus be abrogated.
although the precise mechanism of action is still unknown, it is likely that no igg responses to restv gp <dig>  are induced in the cynomolgus macaques during the acute phase of infection.
since the cynomolgus macaques at the facility where the restv epizootic occurred were euthanized, sequential serum specimens from each cynomolgus macaque were not available.
it is also difficult to determine when each cynomolgus macaque became infected with restv.
we found that three specimens that have anti-gp antibodies were obtained from dead monkeys , however, it is difficult to conclude whether restv infection caused their death because of the possibility of succumbing to infection by shfv or some other agents.
it is predicted that, among the serum samples examined here, nine were acute phase samples because they were positive in ag-elisa or immunohistochemistry
<cit>  .
on the other hand, all but one  of the anti-gp <dig> -positive serum samples were ag negative.
therefore, these cynomolgus macaques were considered to be in the convalescent phase of restv infection.
in this regard, the presence of the anti-gp <dig>  antibody is thought to be a useful indicator for convalescence in cynomolgus macaques infected with restv.
aberrant proinflammatory cytokines/chemokines are a significant factor implicated in the disease progression of ebov- and sudv-infected human cases and experimentally infected cynomolgus macaques
<cit> .
in addition, a balanced proinflammatory response is believed to be a critical factor for determining the disease outcome
<cit> .
we focused on the circulating inflammatory cytokines/chemokines in restv-infected cynomolgus macaques and examined their relationship with convalescence.
we thus found the concentrations of several proinflammatory cytokines/chemokines, such as ifnγ, il <dig>  il- <dig>  and mip1α, to be significantly higher in convalescent sera than in non-convalescent sera.
gupta et al.
recently demonstrated that convalescent serum samples obtained from bdbv-infected human cases include high concentrations of il-1α, il1β, il <dig>  tnfα, and mcp-1
<cit> .
although the exact profiles of proinflammatory cytokines/chemokines shown in our study are different from those reported by gupta et al., these differences are considered most likely to be due to differences among ebolaviruses , host species , and differences related to the disease phase when the samples were obtained.
it is possible that the upregulation of the proinflammatory innate immune responses contributed to the recovery from restv infection in cynomolgus macaques.
in rhesus monkeys experimentally infected with a lethal dose of ebov, anti-inflammatory cytokines, such as il- <dig> and il-1ra, are highly elevated in the acute phase
<cit> .
in human ebola vhf patients, increased concentrations of il- <dig> and il-1ra have been shown in fatal cases, thus suggesting that the mixed anti-inflammatory response syndrome  contributes to the pathogenesis of the hemorrhagic fever caused by ebolaviruses.
since all of the cynomolgus macaques involved in the epizootic were euthanized at the affected facility, the actual fate of the cynomolgus macaques was not clear, and some might have survived the infection.
our data obtained using the sera from cynomolgus macaques in the restv epizootic showed higher il-1ra responses in the convalescent phase than in the non-convalescent phase .
there were no significant differences in the concentration of il- <dig> between the two groups .
this suggests that, unlike other ebolaviruses infections, restv does not induce mars, which is characterized by an elevated induction of il1ra in the acute phase.
in conclusion, we have shown that the anti-gp <dig>  responses, rather than the anti-np responses, in cynomolgus macaques naturally infected with restv were specifically detected in the convalescent stage of restv infection.
in addition, a high concentration of proinflammatory cytokines/chemokines was observed in the convalescent phase.
therefore, the anti-gp <dig>  response and the upregulation of the specific proinflammatory response might be useful indicators of convalescence from restv infection in cynomolgus macaques.
CONCLUSIONS
in this study, we analyzed the humoral responses in cynomolgus macaque serum samples collected during the  <dig> reston outbreak in the philippines and demonstrated that the anti-restv gp <dig>  response and the proinflammatory innate response play significant roles in the convalescence from restv infection in cynomolgus macaques.

 the family filoviridae includes three genera, ebolavirus, marburgvirus, and cuevavirus. the genus ebolavirus currently has five members: bundibugyo virus, ebola virus, reston virus, sudan virus, and taï forest virus. filoviruses induce lethal viral hemorrhagic fevers in both humans and non-human primates, while restv infection in humans is probably subclinical, yet it also causes highly lethal vhf in macaques. restv epizootics among cynomolgus macaques emerged in and in all of these epizootics, the cynomolgus macaques originated in a single primate breeding facility in the philippines. although the natural reservoir of restv remains unknown, restv was isolated from pigs in the philippines, in addition to porcine reproductive and respiratory syndrome virus and porcine circovirus type- in 2008. considering the social impact of ebolaviruses, it is important to understand the endemic and epizootic status of restv in the philippines. in this study, we investigated the antibody responses of cynomolgus macaques that could be dead-end hosts for restv. using serum specimens collected from cynomolgus macaques during a restv outbreak in the philippines in we attempted to elucidate the significance of neutralizing antibodies to restv in viral clearance. we have previously established an enzyme-linked immunosorbent assay and an indirect immunofluorescent antibody assay specific for restv nucleoprotein. these assays are useful tools for investigating the signs of restv infection in cynomolgus macaques. in human cases, antibody responses against ebolaviruses have been analyzed extensively: igg responses to np and other structural proteins have been shown to correlate with asymptomatic and surviving cases, and neutralizing antibody responses targeting the ebolaviruses glycoprotein appear to be the major indicator of protective immunity. on the other hand, proinflammatory cytokines/chemokines are known to play a major role in the pathogenesis of ebolaviruses infections in various species. previous studies have shown an uncontrolled secretion of proinflammatory cytokines/chemokines to contribute to a fatal outcome in ebov-infected humans and cynomolgus macaques. strong proinflammatory cytokine/chemokine responses are also observed in convalescent or asymptomatic cases. in restv-infected cynomolgus macaques, high viremia has been shown to induce the secretion of proinflammatory cytokines/chemokines. however, there have so far only been a limited number of studies on the impact of proinflammatory cytokine/chemokine responses in the convalescent phase of restv infection. in this study, we grouped the cynomolgus macaque samples based on the presence of restv np-antigen in sera and analyzed the antibody reactions and cytokine/chemokine inductions to evaluate the presence of neutralizing antibody to restv. we found that the anti-gp responses, but not the anti-np responses, were closely correlated with the neutralization antibody responses, as well as the clearance of viremia, in the sera of restv-infected cynomolgus macaques. additionally, a high concentration of proinflammatory cytokines/chemokines was detected in the convalescent phase specimens. these data suggest that both the anti-gp responses and proinflammatory cytokines/chemokines play significant roles in the recovery from restv infection in cynomolgus macaques.  restv np-and gp -specific antibodies, neutralizing antibody responses, and the viral antigens in the cynomolgus macaque sera from the restv epizootic twenty-seven serum samples derived from cynomolgus macaques that were either found already dead or had been euthanized at the facility were available. the presence of restv np antigens was evaluated by antigen-capture elisa or immunohistochemistry, while that of anti-restv np igg was evaluated using igg elisa and ifa methods. restv np antigens were detected in the liver in # and while restv np was detected by antigen capture elisa in the sera of # and we therefore assumed that these cynomolgus macaques had suffered from the acute viremic phase of the disease. seventeen of the samples were positive for anti-np igg in igg elisa, while these samples all reacted in ifa. on the other hand, two samples were only positive in ifa. we considered the samples as anti-np igg-positive when either elisa or ifa showed positive reaction. as a result, a total of samples had anti-np igg. cynomolgus macaques with anti-np igg consisted of np antigen-positive and np antigen-negative groups. in order to examine whether the sera contained anti-gp antibodies, we employed a gp -specific elisa and ifa. restv gp Δtm prepared by a baculovirus expression system and restv gp -expressing hela cells were used as antigens for gp -specific elisa and ifa, respectively. anti-restv gp igg were detected in out of the serum samples according to elisa, whereas the remaining samples showed negative reactions. nine serum samples positive for gp antibodies in the igg-elisa also showed positive reactions in the ifa, while one serum sample was only positive in the ifa. serum samples showing positive reactions in either the gp -specific igg-elisa or ifa were considered to be anti-gp positive. interestingly, the sera derived from cynomolgus macaques in the acute viremic phase did not contain any detectable anti-gp igg, although they often contained anti-np igg. *nd: not determined, **euthanized: monkeys were euthanized regardless of clinical manifestation. #nr: not recorded, ag +: antigen positive, ag -: antigen negative, nt+: neutralization antibody positive, nt-: neutralization antibody negative. the specimens with case ids written in italics were considered in acute phase. the specimens with case ids written in bold were considered in convalescent phase. we next attempted to detect the neutralization antibody response in the sera of restv-infected cynomolgus macaques. the vsv pseudotype restv gp was used for the nt assay. twelve serum samples neutralized the vsv-restv-gp /gfp infection, with nt titers ranging from and. the anti-gp igg were not detectable in # and # by igg elisa, while those samples both had a neutralizing activity. these two specimens showed a positive response for viral antigen in the ag-capture elisa and were thus considered to be collected in an early seroconversion phase. all cynomolgus macaques at the facility were euthanatized regardless of clinical status and there was a possibility that some of the cynomolgus macaques had combined infection with simian hemorrhagic fever virus in the animal facility. therefore, immune responses against restv did not always reflect the clinical manifestation. for these reasons, we defined “convalescent” or “non-convalescent” based only upon serological findings. it is noteworthy that, among the serum samples that were positive for viral antigen and negative for the nt antibody, all nine serum samples were negative for anti-gp antibodies, whereas only two samples were negative for anti-np antibodies. on the other hand, all of the ten serum samples that were negative for viral antigen and positive for the nt antibody were positive for both anti-gp and anti-np antibodies. this finding indicates that the anti-gp antibody may therefore increase in cynomolgus macaques in the convalescent phase, while anti-gp antibody is rarely detectable in the acute viremic phase of infection. multiplex assay for cytokines and chemokines in the cynomolgus macaque sera ebola virus infection triggers the expression of several proinflammatory cytokines/chemokines. to examine whether the convalescence from the restv infection correlates with the circulating proinflammatory cytokines/chemokines, eleven restv-infected cynomolgus macaque serum samples were subjected to a multiplex cytokine analysis. since we do not know when the infection occurred for each cynomolgus macaque, it is still unclear whether the sera represented an early or late stage of infection. we used seven convalescent phase sera, and four acute viremic phase sera. among the serum samples, only these serum samples were available for multiplex assay. since the sera were heat-inactivated at 56°c for min prior to being subjected to the multiplex analysis, some cytokines, such as gm-csf and il- which were previously shown to be elevated in some restv infected cynomolgus macaques, could not be measured in the assay. we found that concentrations of several proinflammatory cytokines/chemokines were significantly higher in convalescent than in acute phase sera. this observation is similar to the previous studies showing elevated concentrations of proinflammatory cytokines/chemokines in the convalescent or asymptomatic human cases. in contrast, the concentrations of the five cytokines/chemokines did not differ significantly between the two groups. furthermore, the concentration of mcp- one of the proinflammatory chemokines, was lower in the convalescent than in the non-convalescent sera. these data indicated that ifn-γ, il- il- il-1ra or mip1α might therefore be involved with the host immune responses in the convalescent phase of restv infection. discussion we previously developed a restv np-specific igg-elisa and ifa that proved to be useful for the seroepidemiological studies of cynomolgus macaques during the restv epizootic in the philippines in 1996. the assays based on recombinant np are sensitive for the detection of restv-specific antibodies. on the other hand, anti-gp antibodies are elicited in ebov-infected human cases and are believed to have protective roles against lethal ebov infection. in the present study, in order to gain insight into the igg responses during the recovery from infection with restv, anti-np, anti-gp, and neutralizing antibodies and the level of viremia in the serum specimens were analyzed. the data presented herein showed that the anti-gp response, rather than the anti-np response, was correlated with both the lack in viremia and the neutralizing activities in the sera of restv-infected cynomolgus macaques. there may be at least two possibilities for the lack of anti-gp igg in the acute phase samples. it is known that soluble gp, which does not contain membrane anchor, is secreted during ebolaviruses infection, and it can absorb the anti-gp antibodies. the other possibility is that apoptosis of lymphocytes is induced during restv infection and the resulting host immune responses may thus be abrogated. although the precise mechanism of action is still unknown, it is likely that no igg responses to restv gp are induced in the cynomolgus macaques during the acute phase of infection. since the cynomolgus macaques at the facility where the restv epizootic occurred were euthanized, sequential serum specimens from each cynomolgus macaque were not available. it is also difficult to determine when each cynomolgus macaque became infected with restv. we found that three specimens that have anti-gp antibodies were obtained from dead monkeys, however, it is difficult to conclude whether restv infection caused their death because of the possibility of succumbing to infection by shfv or some other agents. it is predicted that, among the serum samples examined here, nine were acute phase samples because they were positive in ag-elisa or immunohistochemistry. on the other hand, all but one of the anti-gp -positive serum samples were ag negative. therefore, these cynomolgus macaques were considered to be in the convalescent phase of restv infection. in this regard, the presence of the anti-gp antibody is thought to be a useful indicator for convalescence in cynomolgus macaques infected with restv. aberrant proinflammatory cytokines/chemokines are a significant factor implicated in the disease progression of ebov- and sudv-infected human cases and experimentally infected cynomolgus macaques. in addition, a balanced proinflammatory response is believed to be a critical factor for determining the disease outcome. we focused on the circulating inflammatory cytokines/chemokines in restv-infected cynomolgus macaques and examined their relationship with convalescence. we thus found the concentrations of several proinflammatory cytokines/chemokines, such as ifnγ, il il- and mip1α, to be significantly higher in convalescent sera than in non-convalescent sera. gupta et al. recently demonstrated that convalescent serum samples obtained from bdbv-infected human cases include high concentrations of il-1α, il1β, il tnfα, and mcp-1. although the exact profiles of proinflammatory cytokines/chemokines shown in our study are different from those reported by gupta et al., these differences are considered most likely to be due to differences among ebolaviruses, host species, and differences related to the disease phase when the samples were obtained. it is possible that the upregulation of the proinflammatory innate immune responses contributed to the recovery from restv infection in cynomolgus macaques. in rhesus monkeys experimentally infected with a lethal dose of ebov, anti-inflammatory cytokines, such as il- and il-1ra, are highly elevated in the acute phase. in human ebola vhf patients, increased concentrations of il- and il-1ra have been shown in fatal cases, thus suggesting that the mixed anti-inflammatory response syndrome contributes to the pathogenesis of the hemorrhagic fever caused by ebolaviruses. since all of the cynomolgus macaques involved in the epizootic were euthanized at the affected facility, the actual fate of the cynomolgus macaques was not clear, and some might have survived the infection. our data obtained using the sera from cynomolgus macaques in the restv epizootic showed higher il-1ra responses in the convalescent phase than in the non-convalescent phase. there were no significant differences in the concentration of il- between the two groups. this suggests that, unlike other ebolaviruses infections, restv does not induce mars, which is characterized by an elevated induction of il1ra in the acute phase. in conclusion, we have shown that the anti-gp responses, rather than the anti-np responses, in cynomolgus macaques naturally infected with restv were specifically detected in the convalescent stage of restv infection. in addition, a high concentration of proinflammatory cytokines/chemokines was observed in the convalescent phase. therefore, the anti-gp response and the upregulation of the specific proinflammatory response might be useful indicators of convalescence from restv infection in cynomolgus macaques.  in this study, we analyzed the humoral responses in cynomolgus macaque serum samples collected during the reston outbreak in the philippines and demonstrated that the anti-restv gp response and the proinflammatory innate response play significant roles in the convalescence from restv infection in cynomolgus macaques. 
ebolaviruses induce lethal viral hemorrhagic fevers in humans and non-human primates, with the exceptions of reston virus, which is not pathogenic for humans. ebolaebolavirusreston virusreston ebolavirusfiloviruszoonosishumoral immune responsecynomolgus macaquecytokineantibody these results imply that both the antibody response to gp and the proinflammatory innate responses play significant roles in the recovery from restv infection in cynomolgus macaques. in human vhf cases, extensive analyses of the humoral immune responses in survivors and non-survivors have shown that the igg responses to nucleoprotein and other viral proteins are associated with asymptomatic and survival outcomes, and that the neutralizing antibody responses targeting ebolaviruses glycoprotein are the major indicator of protective immunity. on the other hand, the immune responses in non-human primates, especially naturally infected ones, have not yet been elucidated in detail, and the significance of the antibody responses against np and gp in restv-infected cynomolgus macaques is still unclear. in this study, we analyzed the humoral immune responses of cynomolgus macaque by using serum specimens obtained from the restv epizootic in in the philippines to expand our knowledge on the immune responses in naturally restv-infected non-human primates. we found that the anti-gp responses, but not the anti-np responses, closely were correlated with the neutralization responses, as well as the clearance of viremia in the sera of the restv-infected cynomolgus macaques. antigen-capture -elisa was also performed to detect viral antigens in the serum specimens. additionally, by analyzing the cytokine/chemokine concentrations of these serum specimens, we found high concentrations of proinflammatory cytokines/chemokines, such as ifnγ, il il- and mip1α, in the convalescent phase sera. the antibody responses were analyzed using igg-elisa, an indirect immunofluorescent antibody assay, and a pseudotyped vsv-based neutralizing assay.
 ebolaviruses induce lethal viral hemorrhagic fevers in humans and non-human primates, with the exceptions of reston virus, which is not pathogenic for humans. in human vhf cases, extensive analyses of the humoral immune responses in survivors and non-survivors have shown that the igg responses to nucleoprotein and other viral proteins are associated with asymptomatic and survival outcomes, and that the neutralizing antibody responses targeting ebolaviruses glycoprotein are the major indicator of protective immunity. on the other hand, the immune responses in non-human primates, especially naturally infected ones, have not yet been elucidated in detail, and the significance of the antibody responses against np and gp in restv-infected cynomolgus macaques is still unclear. in this study, we analyzed the humoral immune responses of cynomolgus macaque by using serum specimens obtained from the restv epizootic in in the philippines to expand our knowledge on the immune responses in naturally restv-infected non-human primates.  the antibody responses were analyzed using igg-elisa, an indirect immunofluorescent antibody assay, and a pseudotyped vsv-based neutralizing assay. antigen-capture -elisa was also performed to detect viral antigens in the serum specimens. we found that the anti-gp responses, but not the anti-np responses, closely were correlated with the neutralization responses, as well as the clearance of viremia in the sera of the restv-infected cynomolgus macaques. additionally, by analyzing the cytokine/chemokine concentrations of these serum specimens, we found high concentrations of proinflammatory cytokines/chemokines, such as ifnγ, il il- and mip1α, in the convalescent phase sera.  these results imply that both the antibody response to gp and the proinflammatory innate responses play significant roles in the recovery from restv infection in cynomolgus macaques. ebolaebolavirusreston virusreston ebolavirusfiloviruszoonosishumoral immune responsecynomolgus macaquecytokineantibody 