{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,18]],"date-time":"2026-03-18T18:55:04Z","timestamp":1773860104382,"version":"3.50.1"},"reference-count":52,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2022,9,9]],"date-time":"2022-09-09T00:00:00Z","timestamp":1662681600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Biodefense Analysis and Countermeasure","award":["RSRD-05-00378"],"award-info":[{"award-number":["RSRD-05-00378"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>The 2022 global human monkeypox outbreak emphasizes the importance of maintaining poxvirus research, including enriching a basic understanding of animal models for developing and advancing therapeutics and vaccines. Intravenous administration of monkeypox virus in macaques is arguably one of the best animal models for evaluating the efficacy of medical countermeasures. Here we addressed one criticism of the model, a requirement for a high-titer administration of virus, as well as improving our understanding of monkeypox virus pathogenesis. To do so, we infected macaques with a challenge dose containing a characterized inoculum enriched for the extracellular form of monkeypox virus. Although there were some differences between diseases caused by the enriched preparation compared with a relatively similar unpurified preparation, we were unable to reduce the viral input with the enriched preparation and maintain severe disease. We found that inherent factors contained within the serum of nonhuman primate blood affect the stability of the monkeypox extracellular virions. As a first step to study a role of the extracellular form in transmission, we also showed the presence of this form in the oropharyngeal swabs from nonhuman primates exposed to monkeypox virus.<\/jats:p>","DOI":"10.3390\/v14091993","type":"journal-article","created":{"date-parts":[[2022,9,9]],"date-time":"2022-09-09T04:54:41Z","timestamp":1662699281000},"page":"1993","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":15,"title":["Evaluation of Virulence in Cynomolgus Macaques Using a Virus Preparation Enriched for the Extracellular Form of Monkeypox Virus"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4656-5379","authenticated-orcid":false,"given":"Eric M.","family":"Mucker","sequence":"first","affiliation":[{"name":"United States Army Medical Research Institute of Infectious Diseases, Virology Division, Fort Detrick, Frederick, MD 21702, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Josh D.","family":"Shamblin","sequence":"additional","affiliation":[{"name":"United States Army Medical Research Institute of Infectious Diseases, Virology Division, Fort Detrick, Frederick, MD 21702, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Arthur J.","family":"Goff","sequence":"additional","affiliation":[{"name":"United States Army Medical Research Institute of Infectious Diseases, Virology Division, Fort Detrick, Frederick, MD 21702, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Todd M.","family":"Bell","sequence":"additional","affiliation":[{"name":"United States Army Medical Research Institute of Infectious Diseases, Pathology Division, Fort Detrick, Frederick, MD 21702, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Christopher","family":"Reed","sequence":"additional","affiliation":[{"name":"United States Army Medical Research Institute of Infectious Diseases, Pathology Division, Fort Detrick, Frederick, MD 21702, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nancy A.","family":"Twenhafel","sequence":"additional","affiliation":[{"name":"United States Army Medical Research Institute of Infectious Diseases, Pathology Division, Fort Detrick, Frederick, MD 21702, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jennifer","family":"Chapman","sequence":"additional","affiliation":[{"name":"United States Army Medical Research Institute of Infectious Diseases, Pathology Division, Fort Detrick, Frederick, MD 21702, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marc","family":"Mattix","sequence":"additional","affiliation":[{"name":"United States Army Medical Research Institute of Infectious Diseases, Pathology Division, Fort Detrick, Frederick, MD 21702, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Derron","family":"Alves","sequence":"additional","affiliation":[{"name":"National Institutes of Health, National Institute of Allergy and Infectious Diseases, Infectious Disease Pathogenesis Section, Rockville, MD 20852, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5683-3250","authenticated-orcid":false,"given":"Robert F.","family":"Garry","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Immunology, School of Medicine, Tulane University, New Orleans, LA 70112, USA"},{"name":"Zalgen Labs, Frederick, MD 21703, USA"},{"name":"Global Virus Network (GVN), Baltimore, MD 21201, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lisa E.","family":"Hensley","sequence":"additional","affiliation":[{"name":"United States Department of Agriculture, Zoonotic and Emerging Disease Unit, Manhattan, KS 66505, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,9,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1153","DOI":"10.1016\/S1473-3099(22)00228-6","article-title":"Clinical features and management of human monkeypox: A retrospective observational study in the UK","volume":"22","author":"Adler","year":"2022","journal-title":"Lancet Infect. Dis."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"2620","DOI":"10.1128\/AAC.00021-09","article-title":"Nonhuman primates are protected from smallpox virus or monkeypox virus challenges by the antiviral drug ST-246","volume":"53","author":"Huggins","year":"2009","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Mucker, E.M., Chapman, J., Huzella, L.M., Huggins, J.W., Shamblin, J., Robinson, C.G., and Hensley, L.E. (2015). Susceptibility of Marmosets (Callithrix jacchus) to Monkeypox Virus: A Low Dose Prospective Model for Monkeypox and Smallpox Disease. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0131742"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Mucker, E.M., Wollen-Roberts, S.E., Kimmel, A., Shamblin, J., Sampey, D., and Hooper, J.W. (2018). Intranasal monkeypox marmoset model: Prophylactic antibody treatment provides benefit against severe monkeypox virus disease. PLoS Negl. Trop. Dis., 12.","DOI":"10.1371\/journal.pntd.0006581"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"4898","DOI":"10.1128\/JVI.02525-10","article-title":"A novel respiratory model of infection with monkeypox virus in cynomolgus macaques","volume":"85","author":"Goff","year":"2011","journal-title":"J. Virol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"117","DOI":"10.3389\/fcimb.2012.00117","article-title":"Inhalational monkeypox virus infection in cynomolgus macaques","volume":"2","author":"Barnewall","year":"2012","journal-title":"Front. Cell Infect. Microbiol."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Nalca, A., Livingston, V.A., Garza, N.L., Zumbrun, E.E., Frick, O.M., Chapman, J.L., and Hartings, J.M. (2010). Experimental infection of cynomolgus macaques (Macaca fascicularis) with aerosolized monkeypox virus. PLoS ONE, 5.","DOI":"10.1371\/journal.pone.0012880"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1016\/j.virusres.2004.08.015","article-title":"The exit of vaccinia virus from infected cells","volume":"106","author":"Smith","year":"2004","journal-title":"Virus Res."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"42","DOI":"10.1016\/j.virol.2020.02.004","article-title":"Particle-specific neutralizing activity of a monoclonal antibody targeting the poxvirus A33 protein reveals differences between cell associated and extracellular enveloped virions","volume":"544","author":"Mucker","year":"2020","journal-title":"Virology"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"395","DOI":"10.1007\/978-1-4615-5331-1_51","article-title":"Extracellular enveloped vaccinia virus. Entry, egress, and evasion","volume":"440","author":"Smith","year":"1998","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1006\/viro.1998.9516","article-title":"Neutralizing and protective antibodies directed against vaccinia virus envelope antigens","volume":"254","author":"Galmiche","year":"1999","journal-title":"Virology"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"132","DOI":"10.1006\/viro.2000.0750","article-title":"Antibody neutralization of the extracellular enveloped form of vaccinia virus","volume":"280","author":"Law","year":"2001","journal-title":"Virology"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2497","DOI":"10.1091\/mbc.11.7.2497","article-title":"Entry of the two infectious forms of vaccinia virus at the plasma membane is signaling-dependent for the IMV but not the EEV","volume":"11","author":"Locker","year":"2000","journal-title":"Mol. Biol. Cell"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"472","DOI":"10.1016\/j.tim.2008.07.009","article-title":"Vaccinia virus morphogenesis and dissemination","volume":"16","author":"Roberts","year":"2008","journal-title":"Trends Microbiol."},{"key":"ref_15","first-page":"295","article-title":"Protection against poxviruses","volume":"62","author":"Boulter","year":"1969","journal-title":"Proc. R. Soc. Med."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1099\/0022-1317-13-1-9","article-title":"An antigenic difference between intracellular and extracellular rabbitpox virus","volume":"13","author":"Appleyard","year":"1971","journal-title":"J. Gen. Virol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1099\/0022-1317-13-1-19","article-title":"Inactivated smallpox vaccine: Immunogenicity of inactivated intracellular and extracellular vaccinia virus","volume":"13","author":"Turner","year":"1971","journal-title":"J. Gen. Virol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"197","DOI":"10.1099\/0022-1317-23-2-197","article-title":"Neutralizing activities of antisera to poxvirus soluble antigens","volume":"23","author":"Appleyard","year":"1974","journal-title":"J. Gen. Virol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"13139","DOI":"10.1128\/JVI.79.20.13139-13149.2005","article-title":"An orally bioavailable antipoxvirus compound (ST-246) inhibits extracellular virus formation and protects mice from lethal orthopoxvirus Challenge","volume":"79","author":"Yang","year":"2005","journal-title":"J. Virol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1099\/0022-1317-50-1-89","article-title":"Significance of extracellular enveloped virus in the in vitro and in vivo dissemination of vaccinia","volume":"50","author":"Payne","year":"1980","journal-title":"J. Gen. Virol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"4170","DOI":"10.1128\/jvi.66.7.4170-4179.1992","article-title":"Role of cell-associated enveloped vaccinia virus in cell-to-cell spread","volume":"66","author":"Blasco","year":"1992","journal-title":"J. Virol."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Rubins, K.H., Hensley, L.E., Relman, D.A., and Brown, P.O. (2011). Stunned silence: Gene expression programs in human cells infected with monkeypox or vaccinia virus. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0015615"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"6819","DOI":"10.1128\/JVI.03726-13","article-title":"Primary human macrophages serve as vehicles for vaccinia virus replication and dissemination","volume":"88","author":"Byrd","year":"2014","journal-title":"J. Virol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"643","DOI":"10.1099\/0022-1317-66-3-643","article-title":"Extracellular release of enveloped vaccinia virus from mouse nasal epithelial cells in vivo","volume":"66","author":"Payne","year":"1985","journal-title":"J. Gen. Virol."},{"key":"ref_25","first-page":"101","article-title":"Growing poxviruses and determining virus titer","volume":"269","author":"Kotwal","year":"2004","journal-title":"Methods Mol. Biol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2112","DOI":"10.1128\/JVI.01931-10","article-title":"Comparative analysis of monkeypox virus infection of cynomolgus macaques by the intravenous or intrabronchial inoculation route","volume":"85","author":"Johnson","year":"2011","journal-title":"J. Virol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"129","DOI":"10.2217\/fvl.12.130","article-title":"A review of experimental and natural infections of animals with monkeypox virus between 1958 and 2012","volume":"8","author":"Parker","year":"2013","journal-title":"Future Virol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"10889","DOI":"10.1073\/pnas.0804985105","article-title":"Rapid protection in a monkeypox model by a single injection of a replication-deficient vaccinia virus","volume":"105","author":"Earl","year":"2008","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Mucker, E.M., Shamblin, J.D., Raymond, J.L., Twenhafel, N.A., Garry, R.F., and Hensley, L.E. (2022). Effect of Monkeypox Virus Preparation on the Lethality of the Intravenous Cynomolgus Macaque Model. Viruses, 14.","DOI":"10.3390\/v14081741"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Golden, J.W., Josleyn, M., Mucker, E.M., Hung, C.F., Loudon, P.T., Wu, T.C., and Hooper, J.W. (2012). Side-by-side comparison of gene-based smallpox vaccine with MVA in nonhuman primates. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0042353"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"210","DOI":"10.1186\/s12985-017-0880-8","article-title":"Validation of a pan-orthopox real-time PCR assay for the detection and quantification of viral genomes from nonhuman primate blood","volume":"14","author":"Mucker","year":"2017","journal-title":"Virol. J."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"601","DOI":"10.1128\/JCM.42.2.601-609.2004","article-title":"Smallpox and pan-orthopox virus detection by real-time 3\u2032-minor groove binder TaqMan assays on the roche LightCycler and the Cepheid smart Cycler platforms","volume":"42","author":"Kulesh","year":"2004","journal-title":"J. Clin. Microbiol."},{"key":"ref_33","unstructured":"Prophet, E.B., Mills, B., Arrington, J.B., and Sobin, L.H. (1992). Laboratory Methods for Histotechnology, Armed Forces Institute of Pathology."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"478","DOI":"10.1006\/viro.1996.0142","article-title":"Extracellular enveloped vaccinia virus escapes neutralization","volume":"217","author":"Ichihashi","year":"1996","journal-title":"Virology"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1016\/j.virol.2008.04.003","article-title":"Heterogeneity in the A33 protein impacts the cross-protective efficacy of a candidate smallpox DNA vaccine","volume":"377","author":"Golden","year":"2008","journal-title":"Virology"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1006\/viro.1999.9701","article-title":"Production and characterization of human monoclonal antibody Fab fragments to vaccinia virus from a phage-display combinatorial library","volume":"258","author":"Schmaljohn","year":"1999","journal-title":"Virology"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"7046","DOI":"10.1128\/JVI.03158-12","article-title":"The mature virion of ectromelia virus, a pathogenic poxvirus, is capable of intrahepatic spread and can serve as a target for delayed therapy","volume":"87","author":"Ma","year":"2013","journal-title":"J. Virol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"4032","DOI":"10.1128\/jvi.71.5.4032-4041.1997","article-title":"A novel virus binding assay using confocal microscopy: Demonstration that the intracellular and extracellular vaccinia virions bind to different cellular receptors","volume":"71","author":"Vanderplasschen","year":"1997","journal-title":"J. Virol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"7544","DOI":"10.1073\/pnas.95.13.7544","article-title":"Extracellular enveloped vaccinia virus is resistant to complement because of incorporation of host complement control proteins into its envelope","volume":"95","author":"Vanderplasschen","year":"1998","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"5989","DOI":"10.1073\/pnas.0601025103","article-title":"Ligand-induced and nonfusogenic dissolution of a viral membrane","volume":"103","author":"Law","year":"2006","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1201","DOI":"10.1128\/JVI.01797-08","article-title":"Vaccinia virus extracellular enveloped virion neutralization in vitro and protection in vivo depend on complement","volume":"83","author":"Benhnia","year":"2009","journal-title":"J. Virol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1016\/j.virol.2004.07.024","article-title":"Synergistic neutralizing activities of antibodies to outer membrane proteins of the two infectious forms of vaccinia virus in the presence of complement","volume":"328","author":"Lustig","year":"2004","journal-title":"Virology"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1016\/j.virol.2007.06.042","article-title":"Structural basis for the binding of the neutralizing antibody, 7D11, to the poxvirus L1 protein","volume":"368","author":"Su","year":"2007","journal-title":"Virology"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1006\/viro.1995.1378","article-title":"A myristylated membrane protein encoded by the vaccinia virus L1R open reading frame is the target of potent neutralizing monoclonal antibodies","volume":"211","author":"Wolffe","year":"1995","journal-title":"Virology"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"368","DOI":"10.1016\/j.virol.2008.12.019","article-title":"Vaccinia virus L1 binds to cell surfaces and blocks virus entry independently of glycosaminoglycans","volume":"385","author":"Foo","year":"2009","journal-title":"Virology"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"8687","DOI":"10.1128\/JVI.00852-08","article-title":"Vaccinia virus l1 protein is required for cell entry and membrane fusion","volume":"82","author":"Bisht","year":"2008","journal-title":"J. Virol."},{"key":"ref_47","first-page":"479","article-title":"The antibody response in man following infection with viruses of the pox group. III. Antibody response in smallpox","volume":"56","author":"Downie","year":"1958","journal-title":"J. Hyg."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1145","DOI":"10.1017\/S0950268806007618","article-title":"Infectiousness of smallpox relative to disease age: Estimates based on transmission network and incubation period","volume":"135","author":"Nishiura","year":"2007","journal-title":"Epidemiol. Infect."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"8172","DOI":"10.1128\/JVI.00621-10","article-title":"Identification of wild-derived inbred mouse strains highly susceptible to monkeypox virus infection for use as small animal models","volume":"84","author":"Americo","year":"2010","journal-title":"J. Virol."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"174","DOI":"10.1016\/0042-6822(86)90119-4","article-title":"Isolation of cowpox virus A-type inclusions and characterization of their major protein component","volume":"149","author":"Patel","year":"1986","journal-title":"Virology"},{"key":"ref_51","first-page":"670","article-title":"Type A intracytoplasmic inclusions in human cowpox infection","volume":"57","author":"Lawn","year":"2004","journal-title":"J. Clin. Pathol."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"262","DOI":"10.1016\/0042-6822(68)90143-8","article-title":"The relationship between poxvirus and A-type inclusion body during double infection","volume":"36","author":"Ichihashi","year":"1968","journal-title":"Virology"}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/14\/9\/1993\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T00:28:03Z","timestamp":1760142483000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/14\/9\/1993"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,9,9]]},"references-count":52,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2022,9]]}},"alternative-id":["v14091993"],"URL":"https:\/\/doi.org\/10.3390\/v14091993","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,9,9]]}}}