Quantification of Viral and Host Biomarkers in the Liver of Rhesus Macaques

Zaire ebolavirus (EBOV) causes Ebola virus disease (EVD), which carries a fatality rate between 25% and 90% in humans. Liver pathology is a hallmark of terminal EVD; however, little is known about temporal disease progression. We used multiplexed fluorescent immunohistochemistry and in situ hybridization in combination with whole slide imaging and image analysis (IA) to quantitatively characterize temporospatial signatures of viral and host factors as related to EBOV pathogenesis. Eighteen rhesus monkeys euthanized between 3 and 8 days post-infection, and 3 uninfected controls were enrolled in this study. Compared with semiquantitative histomorphologic ordinal scoring, quantitative IA detected subtle and progressive features of early and terminal EVD that was not feasible with routine approaches. Sinusoidal macrophages were the earliest cells to respond to infection, expressing proinflammatory cytokine interleukin 6 (IL6) mRNA, which was subsequently also observed in fibrovascular compartments. The mRNA of interferon-stimulated gene-15 (ISG-15), also known as ISG15 ubiquitin like modifier (ISG15), was observed early, with a progressive and ubiquitous hybridization signature involving mesenchymal and epithelial compartments. ISG-15 mRNA was prominent near infected cells, but not in infected cells, supporting the hypothesis that bystander cells produce a robust interferon gene response. This study contributes to our current understanding of early EVD progression and illustrates the value that digital pathology and quantitative IA serve in infectious disease research. Zaire ebolavirus (EBOV) causes Ebola virus disease (EVD), which carries a fatality rate between 25% and 90% in humans. Liver pathology is a hallmark of terminal EVD; however, little is known about temporal disease progression. We used multiplexed fluorescent immunohistochemistry and in situ hybridization in combination with whole slide imaging and image analysis (IA) to quantitatively characterize temporospatial signatures of viral and host factors as related to EBOV pathogenesis. Eighteen rhesus monkeys euthanized between 3 and 8 days post-infection, and 3 uninfected controls were enrolled in this study. Compared with semiquantitative histomorphologic ordinal scoring, quantitative IA detected subtle and progressive features of early and terminal EVD that was not feasible with routine approaches. Sinusoidal macrophages were the earliest cells to respond to infection, expressing proinflammatory cytokine interleukin 6 (IL6) mRNA, which was subsequently also observed in fibrovascular compartments. The mRNA of interferon-stimulated gene-15 (ISG-15), also known as ISG15 ubiquitin like modifier (ISG15), was observed early, with a progressive and ubiquitous hybridization signature involving mesenchymal and epithelial compartments. ISG-15 mRNA was prominent near infected cells, but not in infected cells, supporting the hypothesis that bystander cells produce a robust interferon gene response. This study contributes to our current understanding of early EVD progression and illustrates the value that digital pathology and quantitative IA serve in infectious disease research. Zaire ebolavirus (EBOV) is a highly infectious and often lethal filovirus that causes hemorrhagic fever with a fatality rate of 25% to 90% in humans.1Malvy D. McElroy A.K. de Clerck H. Gunther S. van Griensven J. Ebola virus disease.Lancet. 2019; 393: 936-948Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 2Baseler L. Chertow D.S. Johnson K.M. Feldmann H. Morens D.M. The pathogenesis of Ebola virus disease.Annu Rev Pathol. 2017; 12: 387-418Crossref PubMed Scopus (182) Google Scholar, 3Zaki S.R. Goldsmith C.S. Pathologic features of filovirus infections in humans.Curr Top Microbiol Immunol. 1999; 235: 97-116PubMed Google Scholar, 4Martines R.B. Ng D.L. Greer P.W. Rollin P.E. Zaki S.R. Tissue and cellular tropism, pathology and pathogenesis of Ebola and Marburg viruses.J Pathol. 2015; 235: 153-174Crossref PubMed Scopus (205) Google Scholar Currently, there are no therapeutics approved by the U.S. Food and Drug Administration for EBOV treatment, though several are being actively evaluated.5Marzi A. Mire C.E. Current Ebola virus vaccine progress.BioDrugs. 2019; 33: 9-14Crossref PubMed Scopus (21) Google Scholar, 6Hoenen T. Groseth A. Feldmann H. Therapeutic strategies to target the Ebola virus life cycle.Nat Rev Microbiol. 2019; 17: 593-606Crossref PubMed Scopus (82) Google Scholar, 7Hayden F.G. Friede M. Bausch D.G. Experimental therapies for Ebola virus disease: what have we learned?.J Infect Dis. 2017; 215: 167-170PubMed Google Scholar Recently, in a landmark achievement, the European Commission (November, 2019) and Food and Drug Administration (December, 2019) granted marketing authorization to Merck's vaccine known as Ervebo (Merck, Kenilworth, NJ), which is a vesicular stomatitis virus vector vaccine system that expresses EBOV glycoprotein (GP). Establishing efficacy of EBOV medical countermeasures in humans is inherently challenging given the unpredictable and rare nature of outbreaks, the diversity and potential for comorbidities in patient populations, as well as unknown inoculation dosage, route, and time of exposure. This emphasizes the importance of utilizing and characterizing highly controlled animal models that best recapitulate our understanding of human disease to allow us to better define the pathobiological signatures of EVD progression. Nonhuman primates (NHPs) represent the gold standard for EBOV animal model research, given their high fidelity to human disease pathophysiology.8Bennett R.S. Huzella L.M. Jahrling P.B. Bollinger L. Olinger Jr., G.G. Hensley L.E. Nonhuman primate models of Ebola virus disease.Curr Top Microbiol Immunol. 2017; 411: 171-193PubMed Google Scholar As in humans, elevated levels of circulating hepatic transaminases and decreased albumin in EBOV-infected NHPs suggest a combination of hepatocyte cytolysis and dysfunction.8Bennett R.S. Huzella L.M. Jahrling P.B. Bollinger L. Olinger Jr., G.G. Hensley L.E. Nonhuman primate models of Ebola virus disease.Curr Top Microbiol Immunol. 2017; 411: 171-193PubMed Google Scholar,9Geisbert T.W. Hensley L.E. Larsen T. Young H.A. Reed D.S. Geisbert J.B. Scott D.P. Kagan E. Jahrling P.B. Davis K.J. Pathogenesis of Ebola hemorrhagic fever in cynomolgus macaques: evidence that dendritic cells are early and sustained targets of infection.Am J Pathol. 2003; 163: 2347-2370Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar Evidence of hepatic involvement and injury as suggested by clinical chemistry values is corroborated histopathologically by hepatocyte necrosis and eosinophilic intracytoplasmic hepatocyte inclusions, which are readily observed in EBOV-infected NHPs by 5 days post-infection (DPI) as well as the rare published human autopsy reports3Zaki S.R. Goldsmith C.S. Pathologic features of filovirus infections in humans.Curr Top Microbiol Immunol. 1999; 235: 97-116PubMed Google Scholar,4Martines R.B. Ng D.L. Greer P.W. Rollin P.E. Zaki S.R. Tissue and cellular tropism, pathology and pathogenesis of Ebola and Marburg viruses.J Pathol. 2015; 235: 153-174Crossref PubMed Scopus (205) Google Scholar EBOV infection also leads to activation of coagulation followed by a consumptive coagulopathy.8Bennett R.S. Huzella L.M. Jahrling P.B. Bollinger L. Olinger Jr., G.G. Hensley L.E. Nonhuman primate models of Ebola virus disease.Curr Top Microbiol Immunol. 2017; 411: 171-193PubMed Google Scholar, 9Geisbert T.W. Hensley L.E. Larsen T. Young H.A. Reed D.S. Geisbert J.B. Scott D.P. Kagan E. Jahrling P.B. Davis K.J. Pathogenesis of Ebola hemorrhagic fever in cynomolgus macaques: evidence that dendritic cells are early and sustained targets of infection.Am J Pathol. 2003; 163: 2347-2370Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 10Geisbert T.W. Young H.A. Jahrling P.B. Davis K.J. Kagan E. Hensley L.E. Mechanisms underlying coagulation abnormalities in Ebola hemorrhagic fever: overexpression of tissue factor in primate monocytes/macrophages is a key event.J Infect Dis. 2003; 188: 1618-1629Crossref PubMed Scopus (320) Google Scholar This is reflected by increased tissue factor (TF) mRNA expression in peripheral blood mononuclear cells (PBMCs) in vitro and in EBOV-infected monkeys, in addition to thrombocytopenia and petechial hemorrhages in NHPs.10Geisbert T.W. Young H.A. Jahrling P.B. Davis K.J. Kagan E. Hensley L.E. Mechanisms underlying coagulation abnormalities in Ebola hemorrhagic fever: overexpression of tissue factor in primate monocytes/macrophages is a key event.J Infect Dis. 2003; 188: 1618-1629Crossref PubMed Scopus (320) Google Scholar Given the integral role the liver plays in EVD, characterizing early events of EBOV replication and dissemination within distinct anatomic compartments/cells and associated host response will serve to enhance our understanding of EVD disease progression and provide benchmarks for evaluating efficacy of medical countermeasures. Promising experimental vaccines and post-exposure medical countermeasures developed using preclinical NHP models have displayed sufficient efficacy to justify advancement into human clinical trials in the 2013 to 2016 Western Africa and ongoing EBOV outbreak in the Democratic Republic of Congo.6Hoenen T. Groseth A. Feldmann H. Therapeutic strategies to target the Ebola virus life cycle.Nat Rev Microbiol. 2019; 17: 593-606Crossref PubMed Scopus (82) Google Scholar The aim of this research was to evaluate the capacity for advanced tissue staining platforms combined with whole-slide imaging and quantitative image analysis (IA) to provide precise morphomolecular benchmarks of early EVD serving to shed light on the pathogenesis of this emerging infectious disease. EBOV strain Kikwit [full designation: Zaire Ebolavirus, H.sapiens-tc/COD/1995/Kikwit-9510621, abbreviated name: EBOV/Kik (https://www.ncbi.nlm.nih.gov/nuccore, accession number MG572235.1)] was obtained from BEI Resources, NIAID, NIH (catalog number NR-50306). A total of 21, 6– to 7–year–old, Chinese origin (4.72 to 10.98 kg) rhesus macaques (8 males and 13 females) were enrolled in this study. Eighteen (n = 18) were inoculated with 1000 plaque-forming units EBOV/Kik in 1 mL of medium/animal via intramuscular injection in the left triceps muscle. Among them, three animals at each time point were humanely euthanized and necropsied at 3 to 6 DPI. Six additional animals were set as terminal disease controls and euthanized upon meeting predetermined clinical endpoint criteria. Three animals served as uninfected controls and were euthanized at 0 DPI. Additional study details not directly pertinent to the results of this paper are being compiled for publication entitled Natural Disease Progression of the Kikwit Ebola Virus Strain in the Rhesus Monkey Animal Model (D.X.L., R.S.B., J.P.L., Donna L. Perry, Timothy K. Cooper, A.M.W.H., John G. Bernbaum, R.J.H., M.A., N.I., William Dowling, Jing Qin, Ian Crozier, Peter B. Jahlring, L.E.H., unpublished data). Tissue samples were fixed for 72 hours in 10% neutral buffered formalin, after which a buffer exchanged was performed. Tissues were removed from the BSL-4 laboratory, processed in a Tissue-Tek VIP-6 automated vacuum infiltration processor (Sakura Finetek USA, Torrance, CA), followed by paraffin with a Tissue-Tek model (Sakura in were to 5 to in and in Tissues were with and for and for the of and analysis was by a of ordinal for and as well as a were for each used to ordinal are in necrosis necrosis necrosis to necrosis of of of in by and cells of and within of and by with of of with and in of of by of and by viral observed viral observed in a immunohistochemistry was using the were and on liver from the and details are in liver were and in 10% for using buffer and a set to to of liver were in for to and were at the and in the followed by of and fluorescent of each was to the and and the in the of were with and with controls served as EBOV terminal animals served as controls were also to of of for and and and viral viral factor coagulation multiplexed fluorescent in a multiplexed fluorescent in situ hybridization was the liver were and with for to The tissue at for followed by in the for at Experimental target and are in were for hours in the at followed by and The rhesus gene (catalog number number was as and the gene of (catalog number number was used as a for and and gene viral fluorescent in situ in a fluorescent in situ were at with the with light and animal tissue which target and were used to for each via on a of were using a to the liver was used to signature that was subsequently removed from whole-slide using were using IA were to tissue fluorescent and to for IA values were in the to and of target and IA was using the and the was used for total of tissue with hybridization on the total tissue as well as total for are on and with the being cellular was the whole-slide for three were and in from each animal to cells 5 from each animal were for analysis using with via with analysis using the in was as Johnson Huzella L. L.M. R.S. Hensley L.E. Jahrling P.B. Olinger G.G. to rhesus macaques from lethal with Ebola virus 2017; PubMed Scopus Google Scholar of liver by was to of with were using the in with the in of and was using experimental BEI in with the were as of viral from liver samples are in was as of tissue factor in from human 2019; PubMed Scopus Google Scholar were from by and to were with factor and factor in the and of and 5 for hours at and the of factor was using a ordinal scoring, the and and for were given the nature of the and given the quantitative and nature of the analysis of and for were performed. were using and for was this the the and are in the of a in with values in The earliest histomorphologic to EVD was observed at 3 by increased of that eosinophilic at were observed and terminal time 5 animals to which with eosinophilic involving and the of of were with and terminal necrosis and liver were were elevated in animals that this as a in EVD with criteria. were observed in animals were observed in animals DPI. A of ordinal are in the and of EBOV infiltration in response to EBOV infection, a fluorescent immunohistochemistry was The EBOV tissue factor (TF) known as and EBOV was to high expression EBOV infection and involvement in of the host interferon of interferon by Ebola and Marburg viruses.J PubMed Scopus Google Scholar was to role in coagulation and in EBOV T.W. Hensley L.E. Larsen T. Young H.A. Reed D.S. Geisbert J.B. Scott D.P. Kagan E. Jahrling P.B. Davis K.J. Pathogenesis of Ebola hemorrhagic fever in cynomolgus macaques: evidence that dendritic cells are early and sustained targets of infection.Am J Pathol. 2003; 163: 2347-2370Abstract Full Text Full Text PDF PubMed Scopus (481) Google T.W. Young H.A. Jahrling P.B. Davis K.J. Kagan E. Hensley L.E. Mechanisms underlying coagulation abnormalities in Ebola hemorrhagic fever: overexpression of tissue factor in primate monocytes/macrophages is a key event.J Infect Dis. 2003; 188: 1618-1629Crossref PubMed Scopus (320) Google Scholar is a highly by and of which are well as cells for EBOV replication and early T.W. Hensley L.E. Larsen T. Young H.A. Reed D.S. Geisbert J.B. Scott D.P. Kagan E. Jahrling P.B. Davis K.J. Pathogenesis of Ebola hemorrhagic fever in cynomolgus macaques: evidence that dendritic cells are early and sustained targets of infection.Am J Pathol. 2003; 163: 2347-2370Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar in and from three and terminal IA results with are in EBOV was observed in in animals at 3 early viral dissemination not observed with and cellular of cells increased at the of total was increased in terminal animals The temporal in of total for in circulating with a = = of EBOV and were increased in animals in of total for and were with = = = = results suggest replication of EBOV in macrophages in the of infected ebolavirus strain Kikwit infection in rhesus macaques results in a temporal in liver monocytes/macrophages with in hepatic tissue factor (TF) and analysis EBOV and analysis EBOV and and are as = animals = 3 of animal and days image the of EBOV infection on infiltration and liver a was used that EBOV a and a hepatocyte which served as of and from three and terminal animals and are in with quantitative IA results in was to 1 results in and as The of total for and increased in animals and was = = total decreased in a and time The in of total was with EBOV and = = = = ebolavirus strain Kikwit infection in rhesus macaques results in a temporal in liver and decreased 1 a in the of analysis EBOV and analysis and EBOV and and EBOV are as = and = 3 of and days image on cellular infiltration to the liver EBOV infection, were also by the of EBOV interferon-stimulated gene (ISG15), and proinflammatory cytokine interleukin 6 (IL6) and of fluorescent are in and with quantitative IA results in EBOV mRNA displayed hybridization of with the to the of viral replication that represent A. M. Feldmann T. Feldmann H. The Ebola virus a PubMed Scopus Google Scholar EBOV mRNA was to with involvement of at and fibrovascular and by 5 DPI. 6 EBOV mRNA within the but prominent in the fibrovascular with time EBOV mRNA expression was increased in animals with time as by quantitative on liver and total of EBOV mRNA the value = = experimental in this ebolavirus strain Kikwit infection in rhesus macaques causes a temporal in the of proinflammatory cytokine interleukin 6 (IL6) and interferon-stimulated also known as ISG15 ubiquitin like modifier (ISG15), within distinct anatomic compartments. 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Hensley L.E. Larsen T. Young H.A. Reed D.S. Geisbert J.B. Scott D.P. Kagan E. Jahrling P.B. Davis K.J. Pathogenesis of Ebola hemorrhagic fever in cynomolgus macaques: evidence that dendritic cells are early and sustained targets of infection.Am J Pathol. 2003; 163: 2347-2370Abstract Full Text Full Text PDF PubMed Scopus (481) Google A.K. R.B. L. C.S. A.K. J.B. Zaki S.R. activation associated with and Ebola virus disease in Infect Dis. 2019; PubMed Scopus Google van de Ebola virus disease features of 2015; PubMed Scopus Google Scholar have also to in from terminal which circulating from the E. response to Ebola virus 2017; Scopus Google Scholar increased total of and in the liver were observed at DPI. Given the of with = = and = = is to suggest that the in the of to the liver is a combination of increased of of which are infected with and with the serving as a of in of to the K. T. of the tissue PubMed Scopus Google Scholar of and EBOV was increased in terminal have not to EBOV suggested to infection and the host response and a role in tissue and of the host M. Olinger G.G. G. D.L. of on on human by Marburg and Ebola viruses.J PubMed Scopus Google Scholar increased viral within viral this of viral within which of and to within hepatic Given have with and activation of this additional for the role in the a and progressive of time hepatocyte necrosis was in the and was not 5 DPI. suggest that in addition to of hepatocyte decreased represent a associated with EVD pathogenesis. targets 1 which is a in the and in H. D. M. H. The for 1 is the PubMed Scopus Google Scholar a rare in humans, to that several clinical that EVD in humans and rate and in J. in the of 1 a 2017; PubMed Scopus Google Scholar our not in EVD, but given that are readily with and and for and patient in with Geisbert T.W. Young H.A. Jahrling P.B. Davis K.J. Kagan E. Hensley L.E. Mechanisms underlying coagulation abnormalities in Ebola hemorrhagic fever: overexpression of tissue factor in primate monocytes/macrophages is a key event.J Infect Dis. 2003; 188: 1618-1629Crossref PubMed Scopus (320) Google Scholar in mRNA and in the peripheral blood of This IA results to the of EBOV and were increased in animals DPI. The the of standard however, temporal of of and by that is on the of cells EBOV EBOV with on the to of a proinflammatory response that results in increased and T.W. Young H.A. Jahrling P.B. Davis K.J. Kagan E. Hensley L.E. Mechanisms underlying coagulation abnormalities in Ebola hemorrhagic fever: overexpression of tissue factor in primate monocytes/macrophages is a key event.J Infect Dis. 2003; 188: 1618-1629Crossref PubMed Scopus (320) Google D.P. S. Ebola virus glycoprotein response in via Microbiol. 2017; PubMed Scopus Google A. A. between Ebola virus glycoprotein and host leads to of proinflammatory and PubMed Scopus Google Scholar The also observed a temporal in levels of in EBOV-infected which by a = = This represent a peripheral blood serving as a for liver analysis of peripheral blood in humans and in NHPs infected with EBOV the expression of interferon-stimulated and proinflammatory as EVD E. C.E. T. E. G. A response to Ebola virus in primates to of PubMed Scopus Google Scholar, E. S. A. D. S. D. J. M. Gunther S. A. signatures from in humans infected with Ebola 2017; PubMed Scopus Google Scholar, E. K. E. C.E. G. of platforms for analysis of whole blood from cynomolgus 2017; PubMed Scopus Google Scholar, M. Hensley L.E. Ebola virus infection leads to a response in circulating 17: PubMed Scopus Google Scholar ISG15 mRNA, to in the blood as early as days of proinflammatory mRNA at of E. S. A. D. S. D. J. M. Gunther S. A. signatures from in humans infected with Ebola 2017; PubMed Scopus Google Scholar levels of in peripheral blood is a hallmark of EVD and is with disease and in NHPs and The role of and in filovirus 2015; PubMed Scopus Google Rollin P.E. and expression in humans infected with Ebola Infect Dis. PubMed Scopus Google Scholar unknown what cellular and their signatures within EBOV-infected are EVD using the were to the expression of host in the of early liver disease. This results suggest there is little expression by with expression from cells, and hepatic suggest that and fibrovascular cells are key to gene expression in the of the liver and represent a peripheral ISG15 mRNA displayed the of total of hybridization of the three and displayed the ISG15 was prominent within and of EBOV was a rare This the hypothesis that EBOV infection of the interferon host bystander cells are a robust interferon response that is in peripheral E. response to Ebola virus 2017; Scopus Google M. Hensley L.E. 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Ebola virus a to PubMed Scopus Google Scholar This provide evidence that IA the potential to and analysis for infectious disease pathogenesis research. of IA with and us to define temporospatial host response to EBOV infection in a highly rhesus model of signatures will serve to models of EVD and in the of efficacy of medical countermeasures. results serve as a for evaluating the of in vitro platforms as human and and platforms to evaluate to recapitulate EVD in animal We the of and for the with from quantitative image analysis analysis using with histomorphologic in the liver of Zaire ebolavirus strain Kikwit rhesus Three days post-infection rare eosinophilic are observed in rare are observed hepatic intracytoplasmic eosinophilic and are with with with with This in of research that are published in the current of The of PDF