the zebov makona strain seed stock originated from serum from a fatal case during the 2014 outbreak in gukdou , guinea ( zebov isolate homo sapiens - wt / gin/2014/ makona - gueckedou - c07 , accession number kj660347.2 ) and was passaged twice in authenticated vero e6 cells obtained from atcc ( atcc , crl-1586 ) [ 11 , 12 ] .
six healthy adult cynomolgus macaques ( macaca fascicularis ) of chinese origin ( weight range , 4.37.0 kg ; age range , 48 years ) were used for these studies . in an initial study , 2 animals were exposed to a target dose of 10 pfu of zebov makona by droplet administration into the medial canthus of each eye , whereas 2 animals were exposed to a target dose of 10 pfu of zebov makona by droplet administration to the oropharynx . in a second study
, 1 animal was exposed to a target dose of 100 pfu of zebov makona by droplet administration into the medial canthus of each eye , while 1 animal was exposed to a target dose of 100 pfu of zebov makona by droplet administration to the oropharynx .
all 6 animals underwent physical examinations , had swab specimens collected from mucosal surfaces , and had blood specimens collected at the time of challenge and on days 2 , 3 , 4 , 5 , 6 , 7 , 8 , 10 , 15 , 21 , and 28 ( for the 10 pfu study ) or 3 , 5 , 7 , 9 , 14 , 21 , and 28 ( for the 100 pfu study ) after virus challenge .
animal studies were completed under biosafety level 4 biocontainment at the galveston national laboratory ( gnl ) and were approved by the university of texas medical branch ( utmb ) institutional laboratory animal care and use committee , in accordance with state and federal statutes and regulations relating to experiments involving animals , and the utmb institutional biosafety committee .
rna was isolated from samples using the viral rnamini kit or rneasy kit ( qiagen ) , using 100 l of blood / swab in 600 l of buffer avl or 100 mg of tissue , respectively , per the manufacturer 's instructions .
primers / probe targeting the vp30 gene of all known zebov strain sequences were used for quantitative reverse transcription polymerase chain reaction ( qrt  pcr ) , with the probe used here being 6-carboxyfluorescein ( 6fam)-59-ccgt caatcaaggagcgcctc39 - 6 carboxytetramethylrhodamine ( tamra ) ( life technologies ) .
zebov rna was detected using the cfx96 detection system ( biorad ) in one - step probe qrt  pcr kits ( qiagen ) , with cycle conditions and genomic rna standard determination as previously described [ 11 , 12 ] .
virus titration was performed by a plaque assay with vero e6 cells obtained from plasma samples as previously described [ 11 , 12 ] . in brief , increasing 10-fold dilutions of the samples were adsorbed to vero e6 monolayers in duplicate wells ( 200 l ) ; the limit of detection was 5 pfu / ml .
total white blood cell counts , white blood cell differentials , red blood cell counts , platelet counts , hematocrit values , total hemoglobin concentrations , mean cell volumes , mean corpuscular volumes , and mean corpuscular hemoglobin concentrations were analyzed in blood specimens collected in tubes containing ethylenediaminetetraacetic acid , using a laser based hematologic analyzer ( beckman coulter ) .
serum samples were tested for concentrations of albumin , amylase , alanine aminotransferase , aspartate aminotransferase , alkaline phosphatase ,  glutamyl transferase , glucose , cholesterol , total protein , total bilirubin , blood urea nitrogen , creatinine , and c - reactive protein by using a piccolo point - of - care analyzer and biochemistry panel plus analyzer discs ( abaxis ) .
tissue samples from major organs were collected for histopathological and ihc examination , immersion fixed in 10% neutral buffered formalin , and processed for histopathologic analysis as previously described [ 11 , 12 ] . for ihc analysis , specific anti - zebov immunoreactivity was detected using an anti - zebov vp40 protein rabbit primary antibody ( integrated biotherapeutics ) at a 1:4000 dilution .
tissue sections were processed for ihc analysis , using the dako autostainer ( dako ) .
secondary antibody used was biotinylated goat anti - rabbit immunoglobulin g ( igg ; vector laboratories ) at 1:200 followed by dako lsab2 streptavidin  horseradish peroxidase ( dako ) .
the zebov makona strain seed stock originated from serum from a fatal case during the 2014 outbreak in gukdou , guinea ( zebov isolate homo sapiens - wt / gin/2014/ makona - gueckedou - c07 , accession number kj660347.2 ) and was passaged twice in authenticated vero e6 cells obtained from atcc ( atcc , crl-1586 ) [ 11 , 12 ] .
six healthy adult cynomolgus macaques ( macaca fascicularis ) of chinese origin ( weight range , 4.37.0 kg ; age range , 48 years ) were used for these studies . in an initial study , 2 animals were exposed to a target dose of 10 pfu of zebov makona by droplet administration into the medial canthus of each eye , whereas 2 animals were exposed to a target dose of 10 pfu of zebov makona by droplet administration to the oropharynx . in a second study
, 1 animal was exposed to a target dose of 100 pfu of zebov makona by droplet administration into the medial canthus of each eye , while 1 animal was exposed to a target dose of 100 pfu of zebov makona by droplet administration to the oropharynx .
all 6 animals underwent physical examinations , had swab specimens collected from mucosal surfaces , and had blood specimens collected at the time of challenge and on days 2 , 3 , 4 , 5 , 6 , 7 , 8 , 10 , 15 , 21 , and 28 ( for the 10 pfu study ) or 3 , 5 , 7 , 9 , 14 , 21 , and 28 ( for the 100 pfu study ) after virus challenge .
animal studies were completed under biosafety level 4 biocontainment at the galveston national laboratory ( gnl ) and were approved by the university of texas medical branch ( utmb ) institutional laboratory animal care and use committee , in accordance with state and federal statutes and regulations relating to experiments involving animals , and the utmb institutional biosafety committee .
rna was isolated from samples using the viral rnamini kit or rneasy kit ( qiagen ) , using 100 l of blood / swab in 600 l of buffer avl or 100 mg of tissue , respectively , per the manufacturer 's instructions .
primers / probe targeting the vp30 gene of all known zebov strain sequences were used for quantitative reverse transcription polymerase chain reaction ( qrt  pcr ) , with the probe used here being 6-carboxyfluorescein ( 6fam)-59-ccgt caatcaaggagcgcctc39 - 6 carboxytetramethylrhodamine ( tamra ) ( life technologies ) .
zebov rna was detected using the cfx96 detection system ( biorad ) in one - step probe qrt 
pcr kits ( qiagen ) , with cycle conditions and genomic rna standard determination as previously described [ 11 , 12 ] .
virus titration was performed by a plaque assay with vero e6 cells obtained from plasma samples as previously described [ 11 , 12 ] . in brief , increasing 10-fold dilutions of the samples were adsorbed to vero e6 monolayers in duplicate wells ( 200 l ) ; the limit of detection was 5 pfu / ml .
total white blood cell counts , white blood cell differentials , red blood cell counts , platelet counts , hematocrit values , total hemoglobin concentrations , mean cell volumes , mean corpuscular volumes , and mean corpuscular hemoglobin concentrations were analyzed in blood specimens collected in tubes containing ethylenediaminetetraacetic acid , using a laser based hematologic analyzer ( beckman coulter ) .
serum samples were tested for concentrations of albumin , amylase , alanine aminotransferase , aspartate aminotransferase , alkaline phosphatase ,  glutamyl transferase , glucose , cholesterol , total protein , total bilirubin , blood urea nitrogen , creatinine , and c - reactive protein by using a piccolo point - of - care analyzer and biochemistry panel plus analyzer discs ( abaxis ) .
tissue samples from major organs were collected for histopathological and ihc examination , immersion fixed in 10% neutral buffered formalin , and processed for histopathologic analysis as previously described [ 11 , 12 ] . for ihc analysis , specific anti - zebov immunoreactivity was detected using an anti - zebov vp40 protein rabbit primary antibody ( integrated biotherapeutics ) at a 1:4000 dilution .
tissue sections were processed for ihc analysis , using the dako autostainer ( dako ) .
secondary antibody used was biotinylated goat anti - rabbit immunoglobulin g ( igg ; vector laboratories ) at 1:200 followed by dako lsab2 streptavidin  horseradish peroxidase ( dako ) .
no study has examined the pathogenicity of the makona strain of zebov in nhps when given at low doses and using routes of exposure more likely to be associated with a natural outbreak . in an initial study
, we exposed 4 cynomolgus macaques to a target dose of 10 pfu by either the oral route ( 2 animals ) or the conjunctival route ( 2 animals ; the actual dose of 10 pfu received was determined by back titration of each inoculum ) . surprisingly
, none of the 4 animals showed any evidence of clinical illness , and there were no changes in any hematologic or serum biochemical parameters during the course of the study ( table 1 ) .
no infectious virus was detected by plaque assay in plasma from any of the 4 animals , and viral rna was below the limit of detection as assessed by qrt - pcr in whole - blood specimens from these animals . with the exception of the nasal swab obtained on day 10 from subject o-2 ( 1.62  10 genome copies / ml ) ,
we were unable to isolate infectious virus from the day 10 nasal swab samples from this animal .
analysis of sera from all 4 animals at days 21 and 28 after exposure showed only low anti - zebov gp elisa igg titers in subject o-1 ( 1:100 at day 21 and 1:50 at day 28 ) .
histopathologic and ihc staining for zebov antigen revealed no significant lesions and no viral antigen present in tissues of any of the animals . 

table 1.clinical description and outcome of zaire ebolavirus ( zebov)challenged nonhuman primatessubject no.sexroutedose , pfuclinical illnessclinical pathologyviremia level , log10 pfu / mlanti - zebov igg titero-1moral10none , survivednone1:100 ( 21 ) ; 1:50 ( 28)o-2foral10none , survivednone0 ( 21 , 28)c-1mconjunctival10none , survivednone0 ( 21 , 28)c-2fconjunctival10none , survivednone0 ( 21 , 28)o-3moral100fever ( d 7 ) , loss of appetite ( d 79 ) , depression ( d 7 , 8) , mild rash ( d 7 , 8) , emesis ( d 7 ) , hematemesis ( d 8) , animal euthanized in the morning of d 8leukocytosis ( d 7 , 8) , thrombocytopenia ( d 7 , 8) , hypoalbuminemia ( d 8) , > 2-fold increase in alt level ( d 7 ) , > 7-fold increase in alt level ( d 8) , >
8-fold increase in ast level ( d 7 ) , > 10-fold increase in ast level ( d 8) , > 4-fold increase in alp level ( d 7 ) , > 7-fold increase in alp level ( d 8) , >
8-fold increase in bun level ( d 8) , > 5-fold increase in cre level ( d 8) , > 3-fold increase in ggt level ( d 7 ) , > 6-fold increase in ggt level ( d 8) , > 4-fold increase in crp level ( d 5 ) , > 10-fold increase in crp level ( d 7 , 8)6.60 ( d5 ) ; 8.75 ( d7 ) ; 8.79 ( d8)ntc-3mconjunctival100loss of appetite ( d 10 ) , survivedlymphopenia ( d 7)1.70 ( d14)1:50 ( 21)days after zebov challenge are in parentheses .
fever is defined as a temperature > 1.4c higher than the baseline level or either 0.7c higher than baseline and 39.7c or 0.6c higher than baseline and 40c .
mild rash is characterized by focal areas of petechiae covering < 10% of the skin .
lymphopenia and thrombocytopenia are defined as a 35% drop in numbers of lymphocytes and platelets , respectively .
leukocytosis is defined by a 2-fold increase in the white blood cell count , compared with baseline .
hypoalbuminemia is defined by a 35% decrease in levels of albumin.abbreviations : alp , alkaline phosphatase ; alt , alanine aminotransferase ; ast , aspartate aminotransferase ; bun , blood urea nitrogen ; cre , creatinine ; crp , c - reactive protein ; ggt ,  glutamyl transferase ; igg , immunoglobulin g ; nt , not tested ; pfu , plaque - forming units . clinical description and outcome of zaire ebolavirus
fever is defined as a temperature > 1.4c higher than the baseline level or either 0.7c higher than baseline and 39.7c or 0.6c higher than baseline and 40c .
mild rash is characterized by focal areas of petechiae covering < 10% of the skin .
lymphopenia and thrombocytopenia are defined as a 35% drop in numbers of lymphocytes and platelets , respectively .
leukocytosis is defined by a 2-fold increase in the white blood cell count , compared with baseline .
abbreviations : alp , alkaline phosphatase ; alt , alanine aminotransferase ; ast , aspartate aminotransferase ; bun , blood urea nitrogen ; cre , creatinine ; crp , c - reactive protein ; ggt ,  glutamyl transferase ; igg , immunoglobulin g ; nt , not tested ; pfu , plaque - forming units .
as it appeared that exposure of cynomolgus macaques to a very low dose of zebov makona by either the oral or conjunctival route did not result in a productive infection causing clinical disease , we performed a second study to determine whether exposure to a slightly higher dose would cause disease in macaques .
two animals were exposed to a target dose of 100 pfu by either the oral route ( 1 animal ) or the conjunctival route ( 1 animal ; actual doses of 120 pfu and 150 pfu were determined by back titration of the oral and conjunctival inocula , respectively ) .
the animal exposed by the oral route ( subject o-3 ) initially showed signs of clinical illness on day 7 , which was characterized by fever , anorexia , and the presence of a macular rash ( table 1 ) .
a high circulating viremia level was noted in subject o-3 beginning at day 5 and increasing at day 8 .
lesions observed by histopathologic staining were consistent with zebov infection and included histiocytosis of axillary and inguinal lymph nodes , lymphocytolysis of white pulp and deposition of fibrin in red pulp of spleen , sinusoidal leukocytosis , rare multifocal necrotizing hepatitis , and numerous pleomorphic intracellular eosinophilic viral inclusion bodies in hepatocytes .
these lesions had correlative strong positive ihc staining for anti - zebov vp40 antigen of mononuclear cells ( macrophages , monocytes , and dendritic ) in red and white pulp of the spleen , sinusoidal spaces of the liver and rare hepatocytes , and mononuclear cells within sinuses of the inguinal and axillary lymph nodes ( figure 1a , 1c , 1e , and 1 g ) .
conversely , the animal exposed by the conjunctival route ( subject c-3 ) did not show any evidence of clinical illness , with the exception of lymphopenia on day 7 and anorexia on day 10 ; this animal survived to the study end point ( table 1 ) .
low - level viremia was detected by plaque assay at day 14 in plasma from subject c-3 ( table 1 ) , while viral rna genomes were detected by qrt - pcr in nasal swabs obtained on days 7 and 9 and the oral swab sample obtained on day 9 . a low anti - zebov gp enzyme - linked immunosorbent assay igg titer ( 1:50 ) was noted in subject c-3 at day 21 .
histopathologic examination of tissues from this animal failed to show lesions that were consistent with zebov infection .
however , weak positive ihc staining for anti - zebov vp40 antigen of mononuclear cells in red pulp of the spleen and in subcapsular and medullary sinuses of the inguinal and axillary lymph nodes ( figure 1b , 1f , and 1h ) was noted at the study end point , although the liver was free of any antigen - positive cells ( figure 1d ) . 

figure 1.comparison of zaire ebolavirus ( zebov ) antigen in representative tissues of cynomolgus monkeys infected via the oral or conjunctival routes .
a , stained spleen tissue section showing strong , diffuse cytoplasmic immunolabeling ( brown ) of dendriform mononuclear cells in the red and white pulp of a zebov - orally infected animals .
b , stained spleen tissue section showing weak , diffuse cytoplasmic immunolabeling of the endothelium and rare dendriform mononuclear cells in the red pulp of a zebov - conjunctivally infected animal .
c , stained liver tissue section showing strong , diffuse cytoplasmic immunolabeling of kupffer cells and rare sinusoidal - lining cells and hepatocytes in a zebov - orally infected animal .
d , stained liver tissue section showing no immunolabeling of zebov - conjunctivally infected animal .
e , stained inguinal lymph node ( ing ln ) section showing strong , diffuse cytoplasmic immunolabeling of dendriform mononuclear cells within the subcapsular and medullary sinuses in a zebov - orally infected animal .
f , stained ing ln section showing very weak , diffuse cytoplasmic immunolabeling of dendriform mononuclear cells within the subcapsular and medullary sinuses in a zebov - conjunctivally infected animal .
g , stained axillary lymph node ( ax ln ) showing strong , diffuse cytoplasmic immunolabeling of dendriform mononuclear cells within the subcapsular and medullary sinuses in a zebov - orally infected animal .
h , stained ax ln showing moderate , diffuse cytoplasmic immunolabeling of dendriform mononuclear cells within the subcapsular and medullary sinuses in a zebov - conjunctivally infected animal .
spleen and liver representative images were taken at 40x original magnification and inguinal and axillary lymph node images at 20x original magnification from orally infected animal o-3 ( a , c , e , and g ) or conjunctivally infected animal c-3 ( b , d , f , and h ) .
comparison of zaire ebolavirus ( zebov ) antigen in representative tissues of cynomolgus monkeys infected via the oral or conjunctival routes .
a , stained spleen tissue section showing strong , diffuse cytoplasmic immunolabeling ( brown ) of dendriform mononuclear cells in the red and white pulp of a zebov - orally infected animals .
b , stained spleen tissue section showing weak , diffuse cytoplasmic immunolabeling of the endothelium and rare dendriform mononuclear cells in the red pulp of a zebov - conjunctivally infected animal .
c , stained liver tissue section showing strong , diffuse cytoplasmic immunolabeling of kupffer cells and rare sinusoidal - lining cells and hepatocytes in a zebov - orally infected animal .
d , stained liver tissue section showing no immunolabeling of zebov - conjunctivally infected animal .
e , stained inguinal lymph node ( ing ln ) section showing strong , diffuse cytoplasmic immunolabeling of dendriform mononuclear cells within the subcapsular and medullary sinuses in a zebov - orally infected animal .
f , stained ing ln section showing very weak , diffuse cytoplasmic immunolabeling of dendriform mononuclear cells within the subcapsular and medullary sinuses in a zebov - conjunctivally infected animal .
g , stained axillary lymph node ( ax ln ) showing strong , diffuse cytoplasmic immunolabeling of dendriform mononuclear cells within the subcapsular and medullary sinuses in a zebov - orally infected animal .
h , stained ax ln showing moderate , diffuse cytoplasmic immunolabeling of dendriform mononuclear cells within the subcapsular and medullary sinuses in a zebov - conjunctivally infected animal .
spleen and liver representative images were taken at 40x original magnification and inguinal and axillary lymph node images at 20x original magnification from orally infected animal o-3 ( a , c , e , and g ) or conjunctivally infected animal c-3 ( b , d , f , and h ) .
previous studies have clearly demonstrated that low doses of the kikwit or mayinga strains of zebov administered to macaques by parenteral injection or small - particle aerosol result in uniformly lethal disease [ 79 ] . additionally , oral or conjunctival exposure of macaques to a high dose of zebov mayinga resulted in severe disease and near uniform lethality .
our results show that low doses ( 10 pfu ) of zebov makona administered by the oral or conjunctival routes resulted in minimal replication of virus and failed to cause a lethal infection .
for example , the dose required to produce a lethal infection may be higher for exposure of conjunctival or pharyngeal mucosal membranes than for parenterally injected virus or small - particle aerosols delivered deep into the lungs .
parenteral exposure simulates an accidental needle stick ; aerosol exposure mimics deliberate release ; and mucosal surface exposure simulates the most likely mode of contact during a natural outbreak .
current knowledge suggests that low - dose exposure from a laboratory accident or intentional release represents a significant risk of lethal zebov infection to any exposed individual , whereas it appears that the threshold of the lethal dose for oral or conjunctival exposure may be at least 10-fold higher than parenteral or aerosol exposure .
while this was a narrowly focused study , our findings are a first step to understanding how much infectious virus is needed for transmission to occur in a natural outbreak . alternatively ,
as has been suggested in other studies , there may be phenotypic differences between the makona strain and the historical mayinga and kikwit strains .
as in our current study , the numbers are still too low with the makona strain to have a completely sound comparison with the mayinga and kikwit strains , but initial steps must be taken to further our understanding .
future studies should focus on determining whether low doses of zebov makona are lethal by the more conventional intramuscular route and by the small - particle aerosol route and to determine whether low doses of other zebov strains are lethal if administered by the oral or conjunctival routes .
 nonhuman primate ( nhp ) models of ebola virus ( ebov ) infection primarily use parenteral or aerosol routes of exposure . 
 uniform lethality can be achieved in these models at low doses of ebov ( 100 plaque - forming units [ pfu ] ) . here , we exposed nhps to low doses of ebov ( makona strain ) by the oral or conjunctival routes . 
 surprisingly , animals exposed to 10 pfu by either route showed no signs of disease . 
 exposure to 100 pfu resulted in illness and/or lethal infection . 
 these results suggest that these more natural routes require higher doses of ebov to produce disease or that there may be differences between makona and historical strains . 
MATERIALS AND METHODS
Challenge Virus
Animal Challenge
Detection of Viremia and Viral RNA
Hematologic and Serum Biochemical Analysis
Histopathologic and Immunohistochemical (IHC) Analyses
RESULTS AND DISCUSSION