From June 5 through 14, 2009, a total of 103 small mammals (trap success rate of 17.6%) were live trapped and sampled from 3 villages in Mali (; ): N’Tessoni (11°2′0′′N, 5°59′0′′W), Soromba (10°35′0′′N, 7°9′0′W), and Doneguebougou (12°48′21′′N, 7°59′0′′W). Pertinent information was recorded for each animal, and ear punch, heart, blood, lung, and liver samples were collected. Mastomys
sp. Rats were the predominant rodent captured (82/103, 79.6%), with all but 4 of these animals identified as M. natalensis
rat by cytochrome B sequence analysis of DNA isolated from ear punch specimens (11
; GenBank accession nos. HM130517–HM130519).
Results of investigation of small mammals trapped in 3 villages in Mali, 2009
Total RNA was extracted from tissue specimens and blood by using RNeasy or QIAamp viral RNA kits (QIAGEN, Valencia, CA, USA), respectively, and screened for the presence of LASV RNA by using a SYBR-green based, real-time reverse transcription–PCR assay that amplifies a 195-bp portion of the small genomic segment (primers Gc656s: 5′-ATTGCTCTTGACTCAGGCC-3′ and Gc851as: 5′-GTGTCCATGTGAATGTGCCTA-3′; TIB Molbiol GBH, Adelphia, NJ, USA). Six infected rodents were identified, each of which had LASV-positive lung, liver, and blood specimens. Positive rodents were genetically identified as M. natalensis, and all were captured from Soromba, for a village prevalence of 24% (6/25; ). Four (66.7%) of 6 infected rodents were male and 5 (83.3%) of 6 were adult. All 6 infected animals were captured indoors.
Tissue homogenates were prepared from selected LASV-positive animals and passaged twice on subconfluent monolayers of Vero E6 cells. After 2 passages, no discernible cytopathic effect was observed, although LASV RNA was detected by reverse transcription–PCR in cells and supernatant. Virus isolation was confirmed by immunoblot analysis in infected cells and supernatant by using a LASV nucleoprotein-specific monoclonal antibody, which detected a 50–55-kDa protein consistent in size with the LASV nucleoprotein (12;
, panel A, www.cdc.gov/EID/content/16/7/1123-appF.htm). Furthermore, electron microscopy performed on infected cells showed viral particles consistent in size and morphologic features with an arenavirus (, panel B).
An ≈800-nt fragment of the LASV polymerase gene was amplified from the isolated virus and the tissues of infected rodents by using a pan–Old World arenavirus assay (13
). Additionally, an 873-bp fragment of the LASV glycoprotein gene, corresponding to the sequence from a British Lassa fever patient (10
), was amplified from the same samples by using primers Gc F1 5′-GCATTTTAATTCAGCCTCAATTAAC-3′ and Gc R1 5′-ATGGGGCAGATTGTGACATTCTTTC-3′. Amplicons were sequenced (GenBank accession nos. GU573541–GU573546 and GU573547–GU573552 for polymerase and glycoprotein fragments, respectively) and aligned with previously described arenavirus sequences by using ClustalX version 2.0.10 software (www.clustal.org
). Nucleotide sequences from the isolated LASV were indistinguishable from the sequences generated from the infected rodent tissues from which they were derived (data not shown). Phylogenetic analysis of the glycoprotein sequences confirmed that all rodent-derived LASV sequences belonged to the same genetic clade as the sequence of the imported Lassa fever case in the United Kingdom. Polymerase fragment sequences confirmed that this clade is most closely related to the previously described AV strain of LASV that originated from the neighboring countries of Côte d’Ivoire, Burkina Faso, or Ghana (14
Figure 2 Phylogenetic analysis of Lassa virus conducted on A) a 754-bp fragment of the polymerase gene (large genomic segment nucleotide positions 3427–4180) and B) a 771-bp fragment of the glycoprotein precursor (small genomic segment nucleotide positions (more ...)