Forensic signatures for Marburgviruses Jian Song a , Murray Wolinsky a , Melinda Wren a , Tom Burr b , Po-E. Li a , Norman Doggett a, * a Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, United States b Computer, Computational, and Statistical Sciences Division, Los Alamos National Laboratory, Los Alamos, NM 87545, United States 1. Introduction Marburg virus was first identified to cause severe cases of hemorrhagic fever in Europe [1]. The epidemic started in mid- August in Marburg, Germany, with three laboratory workers who contracted the disease after processing organs from African green monkeys imported from Uganda. Subsequently, more patients and medical personnel contracted the disease. Six cases also occurred in Frankfurt, Germany, concomitant with the infections in Marburg [2]. In September, two cases were identified in Belgrade, where a veterinarian was infected while performing a necropsy on a dead monkey and transmitted the virus to his wife. A total of 31 cases were identified, with seven fatalities in primary infections (23%). Marburg hemorrhagic fever (MHF) remained an obscure medical curiosity until 1975 when three more cases were reported in Johannesburg, South Africa [2,3]. The index case had traveled to Zimbabwe just prior to his infection and was the only fatality. Two further episodes of MHF were reported in Kenya in 1980 and 1987. The index cases for both the 1980 and 1987 outbreaks had traveled to the Mt. Elgon region, which is located close to Lake Victoria and was the source of the monkeys (trapped near Lake Kyogo, Uganda) that initiated the very first 1967 outbreak [2]. Marburg virus has thus been renamed as Lake Victoria marburgvirus. The first outbreak of MHF in a community setting occurred in 1998 in Durba/Watsa, located in the northeastern region of the Democratic Republic of the Congo (DRC). However, an investiga- tion was not initiated until the death of an attending physician in 1999 [2]. Sporadic cases continued and were directly and indirectly linked to activities in the vicinity of an underground gold mine. Primary cases in this outbreak were gold miners who started multiple, usually short, chains of human-to-human transmission within their families [4]. Overall, 154 cases were reported with 128 fatalities (83%) [5]. Analysis of viral sequences derived from the clinical specimens and virus isolates showed up to 20% sequence diversity. The largest MHF outbreak happened more recently, during 2004–2005 in northern Angola and centered at the city of Uı ´ge. A total of 252 cases were reported with 227 fatalities (91%) during this outbreak [6]. Since this major outbreak, only a small outbreak has occurred in Uganda in 2007, with two cases in young males working in lead and gold mines in Kamwenge district, one of whom died [7]. The most recent case is an imported case of MHF which involved in a Dutch woman who vacationed in Uganda where she was most likely exposed in the Python Cave, which harbors bat species that elsewhere in Africa have been found positive for Marburg virus [8]. Marburgvirus is one of two members of the Filoviridae family, a large group of enveloped viruses, including Ebolavirus, whose Forensic Science International 233 (2013) 338–347 A R T I C L E I N F O Article history: Received 16 July 2013 Accepted 30 September 2013 Available online 9 October 2013 Keywords: Forensic science Hemorrhagic fever virus Marburgvirus Canonical SNPs SNP genotyping TaqMan-MGB allelic discrimination assay A B S T R A C T Marburgvirus is one of the most important hemorrhagic fever viruses with extremely high infectivity and fatality rate (90%). It is transmitted easily in human populations through a respiratory route and therefore considered as a major biothreat agent. Although detection assays have been developed, no assay is available for forensic analysis. Here we report development of forensic assays for Marburgvirus. We performed detailed phylogenetic analysis of strains and isolates from all known Marburg virus outbreaks as well as from several laboratory strains and identified canonical SNPs for all major clades (outbreaks) and strains. TaqMan-MGB allelic discrimination assays targeting these SNPs were designed and experimentally screened against synthetic RNA templates and genomic RNAs. A total of 45 assays were validated to provide 100% coverage of the clades (outbreaks) and 91% at the strain level (21 out of the 23 targeted Marburgvirus strains) with built-in redundancy for increased robustness. Using these validated assays, we were able to provide accurate forensic analysis on 3 ‘‘unknown’’ Marburgviruses. These high-resolution forensic assays allow rapid and accurate genotyping of Marburgviruses for forensic investigations. ß 2013 Elsevier Ireland Ltd. All rights reserved. * Corresponding author. Tel.: +1 505 665 4007; fax: +1 505 665 3024. E-mail address: doggett@lanl.gov (N. Doggett). Contents lists available at ScienceDirect Forensic Science International jou r nal h o mep age: w ww.els evier .co m/lo c ate/fo r sc iin t 0379-0738/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.forsciint.2013.09.026