Ebola Virus Localization in the Macaque Reproductive Tract during Acute Ebola Virus Disease

Abstract

Sexual transmission of Ebola virus (EBOV) has been demonstrated more than a year after recovery from the acute phase of Ebola virus disease (EVD). The mechanisms underlying EBOV persistence and sexual transmission are not currently understood. Using the acute macaque model of EVD, we hypothesized EBOV would infect the reproductive tissues and sought to localize the infection in these tissues using immunohistochemistry and transmission electron microscopy. In four female and eight male macaques that succumbed to EVD between 6 and 9 days after EBOV challenge, we demonstrate widespread EBOV infection of the interstitial tissues and endothelium in the ovary, uterus, testis, seminal vesicle, epididymis, and prostate gland, with minimal associated tissue immune response or organ pathology. Given the widespread involvement of EBOV in the reproductive tracts of both male and female macaques, it is reasonable to surmise that our understanding of the mechanisms underlying sexual transmission of EVD and persistence of EBOV in immune-privileged sites would be facilitated by the development of a nonhuman primate model in which the macaques survived past the acute stage into convalescence. Sexual transmission of Ebola virus (EBOV) has been demonstrated more than a year after recovery from the acute phase of Ebola virus disease (EVD). The mechanisms underlying EBOV persistence and sexual transmission are not currently understood. Using the acute macaque model of EVD, we hypothesized EBOV would infect the reproductive tissues and sought to localize the infection in these tissues using immunohistochemistry and transmission electron microscopy. In four female and eight male macaques that succumbed to EVD between 6 and 9 days after EBOV challenge, we demonstrate widespread EBOV infection of the interstitial tissues and endothelium in the ovary, uterus, testis, seminal vesicle, epididymis, and prostate gland, with minimal associated tissue immune response or organ pathology. Given the widespread involvement of EBOV in the reproductive tracts of both male and female macaques, it is reasonable to surmise that our understanding of the mechanisms underlying sexual transmission of EVD and persistence of EBOV in immune-privileged sites would be facilitated by the development of a nonhuman primate model in which the macaques survived past the acute stage into convalescence. Sexual transmission of filoviruses has been suspected for as long as these viruses have been recognized as human pathogens. During the first recorded outbreak of Marburg virus disease in 1967, sexual transmission from an infected man to a woman was presumed but never proved.1Martini G.A. Marburg virus disease.Postgrad Med J. 1973; 49: 542-546Crossref PubMed Scopus (56) Google Scholar Ebola virus (EBOV) was detected in the semen of a researcher infected while handling diagnostic samples from the first recorded EBOV outbreak in 1976.2Emond R.T. Evans B. Bowen E.T. Lloyd G. A case of Ebola virus infection.Br Med J. 1977; 2: 541-544Crossref PubMed Scopus (262) Google Scholar The 2014 to 2016 EBOV outbreak in West Africa again raised the question of sexual transmission of filoviruses and the potential for asymptomatic infections and EBOV persistence in immune-privileged sites.3Glynn J.R. Bower H. Johnson S. Houlihan C.F. Montesano C. Scott J.T. Semple M.G. Bangura M.S. Kamara A.J. Kamara O. Mansaray S.H. Sesay D. Turay C. Dicks S. Wadoum R.E.G. Colizzi V. Checchi F. Samuel D. Tedder R.S. Asymptomatic infection and unrecognised Ebola virus disease in Ebola-affected households in Sierra Leone: a cross-sectional study using a new non-invasive assay for antibodies to Ebola virus.Lancet Infect Dis. 2017; 17: 645-653Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 4Jacobs M. Rodger A. Bell D.J. Bhagani S. Cropley I. Filipe A. et al.Late Ebola virus relapse causing meningoencephalitis: a case report.Lancet. 2016; 388: 498-503Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 5Sow M.S. Etard J.F. Baize S. Magassouba N.F. Faye O. Msellati P. et al.New evidence of long-lasting persistence of Ebola virus genetic material in semen of survivors.J Infect Dis. 2016; 214: 1475-1476Crossref PubMed Scopus (63) Google Scholar, 6Varkey J.B. Shantha J.G. Crozier I. Kraft C.S. Lyon G.M. Mehta A.K. Kumar G. Smith J.R. Kainulainen M.H. Whitmer S. Stroher U. Uyeki T.M. Ribner B.S. Yeh S. Persistence of Ebola virus in ocular fluid during convalescence.N Engl J Med. 2015; 372: 2423-2427Crossref PubMed Scopus (325) Google Scholar Findings from >17,000 survivors of the outbreak in West Africa suggest sexual transmission is rare. To date, only two recorded incidences of sexual transmission of EBOV from that outbreak have been documented.7Diallo B. Sissoko D. Loman N.J. Bah H.A. Bah H. Worrell M.C. Conde L.S. Sacko R. Mesfin S. Loua A. Kalonda J.K. Erondu N.A. Dahl B.A. Handrick S. Goodfellow I. Meredith L.W. Cotten M. Jah U. Guetiya Wadoum R.E. Rollin P. Magassouba N. Malvy D. Anglaret X. Carroll M.W. Aylward R.B. Djingarey M.H. Diarra A. Formenty P. Keita S. Gunther S. Rambaut A. Duraffour S. Resurgence of Ebola virus disease in Guinea linked to a survivor with virus persistence in seminal fluid for more than 500 days.Clin Infect Dis. 2016; 63: 1353-1356Crossref PubMed Scopus (146) Google Scholar, 8Mate S.E. Kugelman J.R. Nyenswah T.G. Ladner J.T. Wiley M.R. Cordier-Lassalle T. et al.Molecular evidence of sexual transmission of Ebola virus.N Engl J Med. 2015; 373: 2448-2454Crossref PubMed Scopus (309) Google Scholar However, the implications of EBOV transmission by this route are significant given an Ebola virus disease (EVD) mortality rate of >40%.9Crozier I. Ebola virus RNA in the semen of male survivors of Ebola virus disease: the uncertain gravitas of a privileged persistence.J Infect Dis. 2016; 214: 1467-1469Crossref PubMed Scopus (16) Google Scholar, 10Kuhn J.H. Bavari S. Asymptomatic Ebola virus infections: myth or reality?.Lancet Infect Dis. 2017; 17: 570-571Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar We hypothesized that EBOV would infect the reproductive tissues in the macaque model of acute EVD. The purpose of this study was to identify and localize the virus within the reproductive tissues in the macaque model, typically acutely fatal between 6 and 10 days after i.m. challenge. Using this EVD model, we demonstrate EBOV infection of the interstitial or supporting connective tissues of the male and female reproductive organs, with minimal associated tissue immune response or organ pathology. Twelve macaques, seven rhesus (Macaca mulatta; one female and six males) and five cynomolgus (Macaca fascicularis; three females and two males), ranging in age from 35 to 58 months, were challenged with 1000 plaque-forming units i.m. of EBOV Makona C05.11Baize S. Pannetier D. Oestereich L. Rieger T. Koivogui L. Magassouba N.F. Soropogui B. Sow M.S. Keïta S. De Clerck H. Tiffany A. Dominguez G. Loua M. Traoré A. Kolié M. Malano E.R. Heleze E. Bocquin A. Mély S. Raoul H. Caro V. Cadar D. Gabriel M. Pahlmann M. Tappe D. Schmidt-Chanasit J. Impouma B. Diallo A.K. Formenty P. Van Herp M. Günther S. Emergence of Zaire Ebola virus disease in Guinea.N Engl J Med. 2014; 371: 1418-1425Crossref PubMed Scopus (1025) Google Scholar Macaques were euthanized after meeting end point criteria between 6 and 9 days after EBOV challenge. Each macaque underwent a complete postmortem examination when predetermined end point criteria12Warren T.K. Trefry J.C. Marko S.T. Chance T.B. Wells J.B. Pratt W.D. Johnson J.C. Mucker E.M. Norris S.L. Chappell M. Dye J.M. Honko A.N. Euthanasia assessment in ebola virus infected nonhuman primates.Viruses. 2014; 6: 4666-4682Crossref PubMed Scopus (19) Google Scholar for EVD were reached. Reproductive tissues were collected from all macaques for routine histology, immunohistochemistry (IHC) for EBOV viral protein 40 (VP40) matrix protein and/or glycoprotein (GP), and transmission electron microscopy examination.13Noda T. Ebihara H. Muramoto Y. Fujii K. Takada A. Sagara H. Kim J.H. Kida H. Feldmann H. Kawaoka Y. Assembly and budding of Ebolavirus.PLoS Pathog. 2006; 2: e99Crossref PubMed Scopus (133) Google Scholar Immunohistochemistry for EBOV VP40 and/or GP was positive in the reproductive organs of all 12 macaques. Transmission electron microscopy of the reproductive tissues was performed in 11 of 12 macaques that succumbed to EVD, and widespread EBOV infection of the stromal connective tissues, including endothelial cells, was observed in all macaques examined. A summation of findings can be found in Table 1.Table 1Signalment, EBOV in Peripheral Blood, and EBOV Distribution in the Reproductive Tract of Female and Male MacaquesAnimal IDMacaque speciesAge, monthsNecropsy after inoculation, daysEBOV vRNA, copy/mL (log10)EBOV IHC resultsTEM resultsOvaryUterusTestisProstate glandSeminal vesicleEpididymis1Cynomolgus46710.6Stromal/vascularStromal/vascularStromal/EC2Cynomolgus5389.96Stromal/vascularStromal/vascularStromal3Cynomolgus53710.1Stromal/vascularStromal/vascularStromal/EC4Rhesus35710.4Stromal/vascularStromal/vascularStromal5Cynomolgus48610.3Stromal/vascularStromal/vascularStromal/vascularStromal/vascularStromal/EC6Cynomolgus44810Stromal/vascularStromal/vascularStromal/vascularStromal/vascularStromal/EC7Rhesus4498.83Stromal/vascularStromal/vascularStromal/vascularNPStromal/EC8Rhesus4879.78Stromal/vascularStromal/vascularStromal/vascularNPStromal/EC9Rhesus5889.95Stromal/vascularStromal/vascularStromal/vascularStromal/vascularStromal/EC10Rhesus56710.2Stromal/vascularStromal/vascularStromal/vascularStromal/vascularStromal/EC11Rhesus35710.1Stromal/vascularStromal/vascularStromal/vascularStromal/vascularStromal/EC12Rhesus38610.6Stromal/vascularStromal/vascularStromal/vascularStromal/vascularNPEBOV, Ebola virus; EC, endothelial cell; ID, identification; IHC, immunohistochemistry; NP, not performed; TEM, transmission electron microscopy; vRNA, viral RNA. Open table in a new tab EBOV, Ebola virus; EC, endothelial cell; ID, identification; IHC, immunohistochemistry; NP, not performed; TEM, transmission electron microscopy; vRNA, viral RNA. This study was performed in strict adherence to the Guide for the Care and Use of Laboratory Animals14Committee for the Update of the Guide for the Care and Use of Laboratory Animals National Research Council: Guide for the Care and Use of Laboratory Animals.Eighth Edition. National Academies Press, Washington, DC2011Crossref Google Scholar of the NIH, the Office of Animal Welfare, and the US Department of Agriculture. All work was approved by the US National Institute of Allergy and Infectious Diseases, Division of Clinical Research Animal Care and Use Committee, and performed at the US National Institute of Allergy and Infectious Diseases Research Facilities. Procedures were performed after animals had been anesthetized by trained personnel under the supervision of veterinary staff. Food and water were available ad libitum. This study includes 12 macaques that served as controls in three separate studies that were designed to evaluate the efficacy of a therapeutic agent intended to protect against the development of EVD. The first two experiments are described by Johnson et al.15Johnson R.F. Kurup D. Hagen K.R. Fisher C. Keshwara R. Papaneri A. Perry D.L. Cooper K. Jahrling P.B. Wang J.T. ter Meulen J. Wirblich C. Schnell M.J. An inactivated Rabies virus–based Ebola vaccine, FILORAB1, adjuvanted with glucopyranosyl lipid A in stable emulsion confers complete protection in nonhuman primate challenge models.J Infect Dis. 2016; 214: S342-S354Crossref PubMed Scopus (23) Google Scholar The seven rhesus macaques (M. mulatta) and five cynomolgus macaques (M. fascicularis) ranged in age from 35 to 58 months, with a total of four females (three cynomolgus and one rhesus) and eight males (two cynomolgus and six rhesus). These 12 macaques were randomized to control groups before EBOV i.m. challenge with EBOV (Zaire species) in the lateral head of the right triceps muscle using the Makona C05 EBOV isolate at a target dose of 1000 plaque-forming units.11Baize S. Pannetier D. Oestereich L. Rieger T. Koivogui L. Magassouba N.F. Soropogui B. Sow M.S. Keïta S. De Clerck H. Tiffany A. Dominguez G. Loua M. Traoré A. Kolié M. Malano E.R. Heleze E. Bocquin A. Mély S. Raoul H. Caro V. Cadar D. Gabriel M. Pahlmann M. Tappe D. Schmidt-Chanasit J. Impouma B. Diallo A.K. Formenty P. Van Herp M. Günther S. Emergence of Zaire Ebola virus disease in Guinea.N Engl J Med. 2014; 371: 1418-1425Crossref PubMed Scopus (1025) Google Scholar All macaques succumbed to EVD between 6 and 9 days after EBOV challenge. Nine pre-EBOV challenge blood draws were performed in each macaque, the last at 15 days before EBOV challenge (0 day). After EBOV challenge, blood draws were performed every other day starting at 2 days until 8 days after EBOV challenge. The last blood draw was performed immediately before necropsy. Complete blood cell counts, select serum chemistries, and real-time quantitative PCR for viremia were performed on these samples. Whole blood was inactivated in TRIzol LS (Thermo Fisher Scientific, Waltham, MA) in accordance with established methods for inactivation of risk group 4 pathogens. Briefly, total RNA was isolated using the QIAamp Viral RNA Mini Kit (Qiagen, Germantown, MD) using 700 μL of TRIzol LS inactivated sample that was added to 280 μL of Qiagen Buffer AVL containing carrier RNA. The sample was eluted in 70 μL of Buffer AVE, aliquoted, and frozen until assayed. Whole blood viral load was measured using BEI Resources quantitative in accordance with and as viral RNA of Reproductive tissue collected from females and and males seminal and of the at was in in for routine and Ebola virus immunohistochemistry was performed on tissue with an VP40 MD) or GP by a for or for and an Ebola virus was with with and by a controls Ebola from an macaque and from an macaque, as positive and In assay controls of the and controls were performed for MA) and The of EBOV IHC in the reproductive tissues was with VP40 and tissues were for in and in tissues were in and for 2 in After in water and with tissues were in a of and and in were into using a to were collected on and with were in an transmission electron at in the to EVD were not observed in the macaques examined. be in the and for EBOV GP in the was positive in the and and in the endothelium in macaques that succumbed to EVD in the was rare. examination of the viral infection of stromal and endothelial of the or positive for EBOV VP40 and GP not in the to EVD were not observed in the macaques examined. for EBOV VP40 widespread in the stromal cells, and muscle of the in macaques that succumbed to EVD examination of the viral infection of stromal and of the in the were but the of and macaque are and be in the of in M. of of male cynomolgus during and reproductive PubMed Scopus Google Scholar and or L. L. in of semen in PubMed Scopus Google Scholar with of to in of of the Immunohistochemistry for EBOV VP40 and GP in the widespread in the interstitial connective tissues, including endothelial examination of the viral infection of interstitial connective tissues, including endothelial In the prostate and seminal vesicle, to EVD were not widespread for EBOV VP40 and GP in the stromal connective tissues 2 and cell for EBOV by IHC in these organs was rare. In the head and of the epididymis, to EVD were but interstitial widespread for EBOV VP40 and GP was detected in the interstitial connective tissue supporting the of the in acutely macaques examination of the prostate gland, epididymis, and seminal viral infection of interstitial cells, including endothelial 2 and EBOV were within the of an the virus was only observed to from the cell or Ebola virus GP is to in the of infected S. Ebola glycoprotein in the J. PubMed Scopus Google Scholar, N. V. O. C. O. M. A. Ebola virus glycoprotein GP is not when at a 2006; PubMed Scopus Google Scholar which the of observed in the for EBOV VP40 protein was observed not Using the macaque model of acute EVD, we have demonstrated and widespread infection of the male and female reproductive after i.m. EBOV challenge. Ebola virus within the or supporting stromal of the reproductive The of the virus in the and in the macaque model of EVD has not been However, of EBOV in the and IHC associated with in in the have been demonstrated in the model of Jahrling P.B. of Ebola virus infection in Infect Dis. PubMed Scopus (262) Google Scholar The of EBOV infection of on the of and in macaques is The of EBOV infection of muscle in the is in with EVD, an rate of and a rate of have been A. O. P. E. C.S. R.S. S.T. U. Rollin Ebola virus disease in and Infect Dis. 2017; PubMed Scopus Google Scholar Ebola virus is to infect the S. J. Ebola viral disease and Med. 2015; PubMed Scopus Google Scholar In the can be infected with the and A. O. P. E. C.S. R.S. S.T. U. Rollin Ebola virus disease in and Infect Dis. 2017; PubMed Scopus Google Scholar The only infected to EBOV infection was on and 11 days of with the and an of from an EBOV survivor on day 11 of on day of this was for 12 days with the J. C. A. A. De Clerck H. Van Herp M. M.C. V. S. A.K. A. to Ebola virus Infect Dis. 2017; Google Scholar Ebola virus is in and linked transmission from an asymptomatic to a has been D. Keita M. Diallo B. N. D.L. Dahl B.A. J. F. K. S. T. A. V. Faye O. A. Magassouba N. Carroll M.W. Anglaret X. Malvy D. Formenty P. Aylward R. Keita S. Djingarey M. Loman N.J. Gunther S. Duraffour S. Ebola virus persistence in after a of virus transmission from to Infect Dis. 2017; Google Scholar The or of EBOV persistence in is as are the of EVD in and In evidence and of EBOV samples from the 2014 to 2016 West Africa outbreak demonstrated sexual transmission of EBOV to from 6 to year after recovery from B. Sissoko D. Loman N.J. Bah H.A. Bah H. Worrell M.C. Conde L.S. Sacko R. Mesfin S. Loua A. Kalonda J.K. Erondu N.A. Dahl B.A. Handrick S. Goodfellow I. Meredith L.W. Cotten M. Jah U. Guetiya Wadoum R.E. Rollin P. Magassouba N. Malvy D. Anglaret X. Carroll M.W. Aylward R.B. Djingarey M.H. Diarra A. Formenty P. Keita S. Gunther S. Rambaut A. Duraffour S. Resurgence of Ebola virus disease in Guinea linked to a survivor with virus persistence in seminal fluid for more than 500 days.Clin Infect Dis. 2016; 63: 1353-1356Crossref PubMed Scopus (146) Google Scholar, 8Mate S.E. Kugelman J.R. Nyenswah T.G. Ladner J.T. Wiley M.R. Cordier-Lassalle T. et al.Molecular evidence of sexual transmission of Ebola virus.N Engl J Med. 2015; 373: 2448-2454Crossref PubMed Scopus (309) Google Scholar A from et J. A.J. S. E. K. G. S. C. S. K. D. E. K. D. Ebola virus in semen more than two after of acute Ebola virus Infect Dis. 2017; 4 Google Scholar described the between of EVD and the of EBOV RNA in The of was in and was the of our we that persistence is established in the interstitial tissues of the reproductive and tissue are the route by which EBOV is from stromal sites in the seminal vesicle, epididymis, prostate gland, and into the seminal this study tissue collected at from the and examination of seminal fluid was not and tissue was collected for RNA of and of the EBOV but and and cells, for both the and immune are not H. Y. J. J. G. J. F. Ebola viral glycoprotein to 2016; Full Text Full Text PDF PubMed Scopus Google Scholar we have demonstrated EBOV infection of endothelial in reproductive tissues, endothelial a of and endothelial cell infection within one tissue not to of viral infection in M. T. and in development and 2017; PubMed Scopus Google Scholar the macaque model of human EVD is to have a it is the of the virus is in the reproductive of or sexual transmission in human is rare. et R.B. D.L. Rollin and and of Ebola and Marburg 2015; PubMed Scopus Google Scholar for EBOV by IHC in endothelium and within the in one et X. Chance T.B. T.K. Dye J.M. Bavari S. G. J.H. M.G. and of asymptomatic Ebola virus infection in rhesus 2017; 2: PubMed Scopus Google Scholar of EBOV viral RNA in the interstitial tissues of the and epididymis, in the we at day after EBOV in of nonhuman primate EVD survivors after These findings the for an established of EBOV persistence in the macaque model of EVD and to the of viral RNA in the semen to the of The findings from et J. A.J. S. E. K. G. S. C. S. K. D. E. K. D. Ebola virus in semen more than two after of acute Ebola virus Infect Dis. 2017; 4 Google Scholar and et X. Chance T.B. T.K. Dye J.M. Bavari S. G. J.H. M.G. and of asymptomatic Ebola virus infection in rhesus 2017; 2: PubMed Scopus Google Scholar the to evaluate seminal samples from a study for the of EBOV to age the of EBOV EVD to be for efficacy and to the development of EBOV RNA viruses that persistence in infected into EBOV R.E. RNA virus mechanisms and 2017; PubMed Scopus Google Scholar virus infection in after recovery from The stromal of the tissue cells, and in are in the of M. R.F. J.R. B. T. F. E. virus has for stromal and and but not for at the of infection in the reproductive 2017; PubMed Scopus Google Scholar Persistence of virus is associated with in the in seminal fluid for an of days after infection in the of viremia and evidence of reproductive a genetic associated with viral persistence of EBOV in has to be a the of seminal transmission of from EVD has been associated with an and A.K. R.S. Mehta A.K. Kraft C.S. Lyon G.M. Ribner B.S. J. J. Ebola virus infection in immune A. 2015; PubMed Scopus Google Scholar The of EBOV in the or interstitial tissues of the reproductive be one of mechanisms by which the virus in immune-privileged sites to the of of the for the immune cell has been in immune-privileged as the of the in The of the at privileged J 2017; Scopus Google Scholar This is to be in immune of cell The of the at privileged J 2017; Scopus Google Scholar, West K. R. of and in during viral A. PubMed Scopus Google Scholar tissue in the of the an or which the development of viral M. M. H. M. G. L. H. L. T. A. S. of the of the that the and of 2017; PubMed Scopus Google Scholar The virus the immune including the of two and that the of in cells, and T. S. The Ebola virus and has a Pathog. PubMed Scopus Google Scholar, A. P. B. M. M. A. R. R. J. Magassouba N. Gabriel M. Gunther S. Oestereich L. C. Ebola virus disease is by and of Infect Dis. 2016; 214: PubMed Scopus Google Scholar During the virus GP while a GP that antibodies against EBOV GP on cell L. C. a of immune by Ebola Pathog. PubMed Scopus Google Scholar In EBOV infections the of P. M.C. S.T. Ebola virus and PubMed Scopus Google Scholar, P. L. D. and Ebola virus 2016; 388: Full Text Full Text PDF PubMed Scopus Google Scholar A nonhuman primate EBOV model would be to study the mechanisms that the development of EBOV persistence in immune-privileged sites and to this virus to in We and for with tissue and the immunohistochemistry on the tissues for with the of the Cooper and for the E. and the Viral and Research for to these