title: Monkeypox virus detection in rodents using real-time 3'-minor groove binder TaqMan assays on the Roche LightCycler.
abstract: During the summer of 2003, an outbreak of human monkeypox occurred in the Midwest region of the United States. In all, 52 rodents suspected of being infected with monkeypox virus were collected from an exotic pet dealer and from private homes. The rodents were euthanized and submitted for testing to the United States Army Medical Research Institute of Infectious Diseases by the Galesburg Animal Disease Laboratory, Illinois Department of Agriculture. The rodent tissue samples were appropriately processed and then tested by using an integrated approach involving real-time polymerase chain reaction (PCR) assays, an antigen-detection immunoassay, and virus culture. We designed and extensively tested two specific real-time PCR assays for rapidly detecting monkeypox virus DNA using the Vaccinia virus F3L and N3R genes as targets. The assays were validated against panels of orthopox viral and miscellaneous bacterial DNAs. A pan-orthopox electrochemiluminescence (ECL) assay was used to further confirm the presence of Orthopoxvirus infection of the rodents. Seven of 12 (58%) animals (seven of 52 (15%) of all animals) tested positive in both monkeypox-specific PCR assays and two additional pan-orthopox PCR assays (in at least one tissue). The ECL results showed varying degrees of agreement with PCR. One hamster and three gerbils were positive by both PCR and ECL for all tissues tested. In addition, we attempted to verify the presence of monkeypox virus by culture on multiple cell lines, by immunohistology, and by electron microscopy, with negative results. Sequencing the PCR products from the samples indicated 100% identity with monkeypox virus strain Zaire-96-I-16 (a human isolate from the Congo). These real-time PCR and ECL assays represent a significant addition to the battery of tests for the detection of various orthopoxviruses. In light of the recent monkeypox virus transmissions, early detection of the virus is crucial for both natural outbreaks and potential acts of bioterrorism.
authors.affiliation: Diagnostic Systems Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, USA. David.Kulesh@amedd.army.mil
authors.forename: David A
authors.initials: DA
authors.lastname: Kulesh
authors.forename: Bonnie M
authors.initials: BM
authors.lastname: Loveless
authors.forename: David
authors.initials: D
authors.lastname: Norwood
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Garrison
authors.forename: Chris A
authors.initials: CA
authors.lastname: Whitehouse
authors.forename: Chris
authors.initials: C
authors.lastname: Hartmann
authors.forename: Eric
authors.initials: E
authors.lastname: Mucker
authors.forename: David
authors.initials: D
authors.lastname: Miller
authors.forename: Leonard P
authors.initials: LP
authors.lastname: Wasieloski
authors.forename: John
authors.initials: J
authors.lastname: Huggins
authors.forename: Gregory
authors.initials: G
authors.lastname: Huhn
authors.forename: Lori L
authors.initials: LL
authors.lastname: Miser
authors.forename: Carroll
authors.initials: C
authors.lastname: Imig
authors.forename: Mark
authors.initials: M
authors.lastname: Martinez
authors.forename: Tom
authors.initials: T
authors.lastname: Larsen
authors.forename: Cynthia A
authors.initials: CA
authors.lastname: Rossi
authors.forename: George V
authors.initials: GV
authors.lastname: Ludwig
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods / Q000662:veterinary*; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D004196:Disease Outbreaks / Q000662:veterinary*; D004563:Electrochemistry; D007087:Illinois / Q000453:epidemiology; D008163:Luminescent Measurements; D045908:Mpox, Monkeypox / Q000175:diagnosis / Q000453:epidemiology / Q000662:veterinary* / Q000821:virology; D008993:Monkeypox virus / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods / Q000662:veterinary*; D012376:Rodent Diseases / Q000175:diagnosis* / Q000453:epidemiology / Q000821:virology; D019914:Taq Polymerase
chemical_list: D004279:DNA, Viral; D019914:Taq Polymerase