New Lineage of Lassa Virus, Togo, 2016.
Lassa virus has not been previously detected in humans or rodents in Togo; therefore, the virus was not considered endemic to this country. We describe a strain of Lassa virus representing a new lineage that was isolated from a cluster of human infections with an epidemiologic link to Togo (online Technical Appendix, https://wwwnc.cdc.gov/ EID/article/24/3/17-1905-Techapp1.pdf) (6,7). The clinical courses of the 3 case-patients and medical and public health interventions are described elsewhere (8-10).
The Lassa virus infections in the index case-patient, secondary case-patient 1, and secondary case-patient 2 were confirmed by laboratory investigations at Bernhard Nocht Institute (Hamburg, Germany); Centers for Disease Control and Prevention (Atlanta, GA, USA); and Philipps University (Marburg, Germany), respectively. The viruses from all 3 patients were isolated in Vero E6 cell culture in the respective Biosafety Level 4 laboratories. Full-length virus sequences were generated directly from clinical specimens, from the isolates, or both using next-generation sequencing technology in combination with Sanger sequencing (sequences deposited into GenBank under accession nos. KU961971, KU961972, LT601601, LT601602, MF990886MF990889) (online Technical Appendix). The sequence from the index case-patient was submitted to GenBank on March 23, 2016, and immediately made publicly available to support the laboratory and public health response in Togo and the other affected countries.
The virtually identical viruses from the 3 patients confirmed the transmission chains suggested by the epidemiologic data. Only the virus from secondary case-patient 2 showed differences in coding regions--a deletion of 3 nt and a nucleotide exchange in the polymerase (L) gene--from the viruses in the other 2 case-patients. These differences were confirmed by sequencing the virus in the clinical specimens. Differences among the 3 strains in the highly structured intergenic regions might represent artifacts created by the difficulty in sequencing these regions.
The phylogeny was inferred using BEAST2 (https:// www.beast2.org/) with nucleotide sequences of full-length nucleoprotein (NP), glycoprotein precursor (GPC), and L and Z genes of the Togo strain in conjunction with representative sequences of Lassa virus and other Old World arenaviruses. The most stable reconstruction was obtained for the L gene with the Togo strain being placed in sister relationship with lineage II (all branches with posterior support values >0.97) (Figure). In the NP- and GPC-based phylogenies, the Togo strain clusters with lineages I and VI (Pinneo and Kako strains); however, the branching order is not well supported (posterior values 0.51-0.86) (Figure). The phylogeny based on the small Z gene further supports a relationship of the Togo strain with lineages I, II, and VI (online Technical Appendix Figure 1). The ambiguous position of the Togo strain relative to lineages I, II, and VI is consistent with a recombination analysis showing that most of the L gene sequence is related to lineage II, and NP and GPC comprise sequence stretches mainly related to lineages I and VI (online Technical Appendix Figure 2). This mosaic structure might be the result of recombination, reassortment, or both or might have evolved by chance.
The long branch (i.e., large phylogenetic distance) separating the Togo strain from known lineages suggests that it represents a new lineage. Because Lassa virus lineages were originally established on the basis of uncorrected sequence distances (1), we used the same method here. The frequency distribution of pairwise amino acid distances in GPC, NP, and L between the Togo strain and all other Lassa virus strains perfectly overlaps with the distribution of distances between Lassa virus lineages I, II, III, IV, and VI indicating that the Togo strain is a separate lineage (online Technical Appendix Table 1). However, we noted that the distance between the proposed lineage V and lineage IV rather corresponds to intralineage distances, and therefore, we considered lineage V a subclade of lineage IV in our analysis (online Technical Appendix Table 1). We propose that formal recognition of Lassa virus lineages should be decided by the International Committee on Taxonomy of Viruses.
In conclusion, sequencing Lassa virus from a cluster of imported infections, with the index case-patient originating from Togo, reveals a new lineage of Lassa virus in West Africa. It seems to be related to lineage II or lineages I/VI, which are all circulating in Nigeria.
We thank Debi Cannon, Cheng-Feng Chiang, Aridth Gibbons, James Graziano, Mary Jenks, Maria Morales-Betouille, Nishi Patel, Alexander Schlaphof, Corinna Thome-Bolduan, Neele Neddersen, and Sabine Kleuckling for their assistance in performing the laboratory testing and Gotthard Ludwig and Michael Schmidt for their technical assistance with the Biosafety Level 4 facility at Philipps University.
This study was supported by the German Research Foundation (grants SFB 1021 TP A02/B03 to S.B., TP B05 to T.S., and TP Z02 to T.H.; GU 883/4-1 to S.G.; and FI1787/2-1 to E.F.C.) and the European Commission through the Horizon 2020 project EVAg (European Virus Archive goes global), grant agreement no. 653316. We also received support from the Serious Communicable Diseases Program at Emory Hospital, which is supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under award no. UL1TR000454.
Dr. Whitmer is a microbiologist in CDC's Viral Special Pathogens Branch, Division of High-Consequence Pathogens and Pathology, National Center for Emerging and Zoonotic Infectious Diseases. Her research interests include sequencing of RNA viruses, viral pathogen discovery, bioinformatics, virus evolution, and molecular epidemiology.
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Address for correspondence: Stephan Gunther, Department of Virology, Bernhard-Nocht-Institute for Tropical Medicine, Bernhard-Nocht-Str. 74, 20359 Hamburg, Germany; email: email@example.com
Shannon L.M. Whitmer,  Thomas Strecker,  Daniel Cadar,  Hans-Peter Dienes, Kelly Faber, Ketan Patel, Shelley M. Brown, William G. Davis, John D. Klena, Pierre E. Rollin, Jonas Schmidt-Chanasit, Elisabeth Fichet-Calvet, Bernd Noack, Petra Emmerich, Toni Rieger, Svenja Wolff, Sarah Katharina Fehling, Markus Eickmann, Jan Philipp Mengel, Tilman Schultze, Torsten Hain, William Ampofo, Kofi Bonney, Juliana Naa Dedei Aryeequaye, Bruce Ribner, Jay B. Varkey, Aneesh K. Mehta, G. Marshall Lyon III, Gerrit Kann, Philipp De Leuw, Gundolf Schuettfort, Christoph Stephan, Ulrike Wieland, Jochen W.U. Fries, Matthias Kochanek, Colleen S. Kraft, Timo Wolf, Stuart T. Nichol, Stephan Becker,  Ute Stroher,  Stephan Gunther 
Author affiliations: Centers for Disease Control and Prevention, Atlanta, Georgia, USA (S.L.M. Whitmer, K. Patel, S.M. Brown, W.G. Davis, J.D. Klena, P.E. Rollin, S.T. Nichol, U. Stroher); Philipps University, Marburg, Germany (T. Strecker, S. Wolff, S.K. Fehling, M. Eickmann, S. Becker); German Center for Infection Research (DZIF), Partner sites, Hamburg, Giessen, and Marburg, Germany (T. Strecker, S. Wolff, S.K. Fehling, M. Eickmann, T. Hain, S. Becker, S. Gunther); Bernhard Nocht Institute for Tropical Medicine, Hamburg (D. Cadar, J. Schmidt-Chanasit, E. Fichet-Calvet, B. Noack, P. Emmerich, T. Rieger, S. Gunther); University Hospital of Cologne, Cologne, Germany (H.-P. Dienes, U. Wieland, J.W.U. Fries, M. Kochanek); Hospital of Hope, Mango, Togo (K. Faber); University of Rostock, Rostock, Germany (P. Emmerich); Justus-Liebig University Giessen, Giessen (J.P. Mengel, T. Schultze, T. Hain); Noguchi Memorial Institute for Medical Research, Accra, Ghana (W. Ampofo, K. Bonney, J.N.D. Aryeequaye); Emory University, Atlanta (B. Ribner, J.B. Varkey, A.K. Mehta, G.M. Lyon III, C.S. Kraft); University Hospital, Frankfurt/Main, Germany (G. Kann, P. De Leuw, G. Schuettfort, C. Stephan, T. Wolf)
 These first authors contributed equally to this article.
 These senior authors contributed equally to this article.
Caption: Figure. Phylogeny of the Lassa virus strain from Togo, 2016. Phylogenetic trees were inferred by using BEAST2 (https://www.beast2. org/) for full-length glycoprotein precursor (A), nucleoprotein (B), and polymerase (C) genes. The analysis included representative Lassa virus strains and other Old World arenaviruses (online Technical Appendix, https://wwwnc.cdc.gov/EID/article/24/3/17-1905Techapp1.pdf). Posterior support values are shown at the branches. Lassa virus lineages are indicated by roman numbers on the right. The branch for Mopeia and Mobala virus is shown schematically and the branches for the remaining Old World arenaviruses have been removed for clarity of presentation. The branches for the Togo strain and most closely related Lassa virus lineages are labeled ([dagger], Togo; ([double dagger]), lineage II; *, lineages I and VI). The origins of the Lassa virus strains are abbreviated as follows: CIV, Cote d'Ivoire; GUI, Guinea; LIB, Liberia; MAL, Mali; NIG, Nigeria; SL, Sierra Leone. A color version of this figure is available online (https://wwwnc.cdc.gov/EID/article/24/3/17-1905-F1.htm).
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|Publication:||Emerging Infectious Diseases|
|Date:||Mar 1, 2018|
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