Detection of Lassa Virus, Mali

To determine whether Lassa virus was circulating in southern Mali, we tested samples from small mammals from 3 villages, including Soromba, where in 2009 a British citizen probably contracted a lethal Lassa virus infection. We report the isolation and genetic characterization of Lassa virus from an area previously unknown for Lassa fever.

L assa fever is an acute viral infection associated with a wide spectrum of disease manifestations, which range from mild to hemorrhagic fever characterized by multiorgan failure (1). The etiologic agent of Lassa fever is Lassa virus (LASV, family Arenaviridae, genus Arenavirus), which is maintained in its natural rodent reservoir, the multimammate rat (Mastomys natalensis) (1)(2)(3). Although M. natalensis rats are ubiquitous in many parts of sub-Saharan Africa, infected rodents have only been reported in West African countries, most commonly Nigeria, Sierra Leone, and Guinea (3)(4)(5). Consequently, cases of Lassa fever mainly occur in regions in which the virus is endemic, consisting of those 3 countries and Liberia, with an annual incidence ranging from 300,000 to 500,000 cases and ≈5,000 deaths (6). Serologic evidence of LASV infections has also been reported from other West and Central African countries (5,(7)(8)(9). Additionally, LASV has been introduced into Europe and North America several times, making Lassa fever one of the most prominent imported exotic viral hemorrhagic fevers, which strongly affects public health systems (9).
In 2009, Lassa fever was diagnosed postmortem in a young man with a 10-day history of fever who had been evacuated from Mali to London (10). The patient had no travel history to any LASV-endemic region, which suggests that he contracted the infection in Mali, most likely while working in the village of Soromba (Figure 1). In this report, we provide evidence that LASV is circulating in M. natalensis in southern Mali, thereby expanding the geo-graphic distribution of LASV in West Africa and posing a risk to humans in an area previously unknown for Lassa fever. The study was carried out in accordance with a protocol approved by an Institutional Animal Care and Use Committee of the US National Institutes of Health.
Total RNA was extracted from tissue specimens and blood by using RNeasy or QIAamp viral RNA kits (QIA-GEN, 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 amplifi es 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 identifi ed, each of which had LASV-positive lung, liver, and blood specimens. Positive rodents were genetically identifi ed as M. natalensis, and all were captured from Soromba, for a village prevalence of 24% (6/25, Table). 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 subconfl uent 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 confi rmed by immunoblot analysis in infected cells and supernatant by using a LASV nucleoprotein-specifi c monoclonal antibody, which detected a 50-55-kDa protein consistent in size with the LASV nucleoprotein (12; online Appendix Figure, 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 (online Appendix Figure, panel B).
An ≈800-nt fragment of the LASV polymerase gene was amplifi ed 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 amplifi ed 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 Clust-alX 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 confi rmed 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 confi rmed 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).

Conclusions
This study demonstrates that M. natalensis rats in Mali carry a genetically unique strain of LASV (proposed name Soromba-R). Genetically, Soromba-R is nearly indistinguishable from the nucleotide sequence generated from a lethal case of Lassa fever imported from Mali to the United Kingdom, which supports the epidemiologic data that the infection occurred in the village of Soromba in southern Mali (10). The Soromba-R strain of LASV is genetically closely related to the AV strain, which was isolated from an imported lethal Lassa fever case in a patient who contracted the infection while traveling in neighboring countries (14). Thus, this study provides evidence for an expanded region of LASV endemicity in West Africa.
On the basis of Mali's geographic proximity to countries where LASV is endemic, it should not be surprising that LASV is circulating there. Cases of Lassa fever are diagnosed annually in Guinea, and sporadic cases have been diagnosed in Burkina Faso and Côte d'Ivoire, all of which border Mali (5,14,15). Furthermore, although to date only 1 laboratory-confi rmed Lassa fever case has been diagnosed from Mali, there is serologic evidence of a LASV infection in 1971 in a missionary stationed in Mali (8). Considering this probable case and the identifi cation of a genetically unique strain of LASV in rodents and a human case from Mali, it is likely that LASV has been present in Mali yet undetected for decades. The nonspecifi c clinical signs and symptoms of a large proportion of LASV infections, combined with the unfamiliarity of Malian physicians with Lassa fever, suggest that the infection could easily be misdiagnosed. Currently, Lassa fever is not considered in the differential diagnosis for febrile illness in Mali. This report, however, provides evidence of an emerging infectious disease problem with public health effects; thus, Lassa fever diagnostics and surveillance should be implemented at least in the southern regions of Mali.
Soromba and the surrounding area may represent an enzootic hotspot for LASV-infected rodents. However, a greater distribution of LASV in Mali should not be ruled out, particularly in the southern regions of the country where the climate and geography are similar to conditions found in many parts of Guinea and Sierra Leone ( Figure   DISPATCHES 1124 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 7, July 2010 1). Combined with our fi ndings that M. natalensis was the predominant species of rodent captured in all 3 villages in Mali suggests that conditions favor LASV circulation. Furthermore, on the basis of the timing of the peak incidence of Lassa fever cases, which typically corresponds with the dry season (15), we believe that the proportion of LASVinfected rodents may have been low at the time of these studies and beyond the sensitivity of the limited sampling effort conducted here. Future studies need to address these concerns as well as determine the geographic distribution of LASV-infected rodents in Mali to help focus public health preparedness efforts.