The 2018 Nigerian Lassa fever season saw the largest ever recorded upsurge of cases, raising concerns over the emergence of a strain with increased transmission rate. To understand the molecular epidemiology of this upsurge we performed, for the first time at the epicenter of an unfolding outbreak, metagenomic nanopore sequencing directly from patient samples, an approach dictated by the highly variable genome of the target pathogen. Genomic data and phylogenetic reconstructions were communicated immediately to Nigerian authorities and the WHO to inform the public health response. Real-time analysis of 36 genomes, and subsequent confirmation using all 120 samples sequenced in-country, revealed extensive diversity and phylogenetic intermingling with strains from previous years, suggesting independent zoonotic transmission events; allaying concerns of an emergent strain or extensive human-to-human transmission.

Targeting of peroxisomal membrane proteins (PMPs) is a multistep process that requires not only recognition of PMPs in the cytosol but also their insertion into the peroxisomal membrane. As a consequence, targeting signals of PMPs (mPTS) are rather complex. A candidate protein for the PMP recognition event is Pex19p, which interacts with most PMPs. However, the respective Pex19p-binding sites are ill-defined and it is currently disputed whether these sites are contained within mPTS. By using synthetic peptide scans and yeast two-hybrid analyses, we determined and characterized Pex19p-binding sites in Pex11p and Pex13p, two PMPs from Saccharomyces cerevisiae. The sites turned out to be composed of a short helical motif with a minimal length of 11 amino acids. With the acquired data, it proved possible to predict and experimentally verify Pex19p-binding sites in several other PMPs by applying a pattern search and a prediction matrix. A peroxisomally targeted Pex13p fragment became mislocalized to the endoplasmic reticulum in the absence of its Pex19p-binding site. By adding the heterologous binding site of Pex11p, peroxisomal targeting of the Pex13p fragment was restored. We conclude that Pex19p-binding sites are well-defined entities that represent an essential part of the mPTS. INTRODUCTIONPeroxisomes are ubiquitous organelles of eukaryotic cells, whose proteins are imported posttranslationally. Matrix proteins are directed to peroxisomes by either of two targeting signals, a C-terminal PTS1 or an N-terminal PTS2. The topogenesis of peroxisomal membrane proteins (PMPs) is accomplished by yet another mechanism, because most of the peroxin mutants, characterized by their defect in the biogenesis of peroxisomes, exhibit a block in matrix protein import, but do import PMPs normally (Lazarow and Fujiki, 1985;Gould and Valle, 2000;Subramani et al., 2000;Purdue and Lazarow, 2001;Eckert and Erdmann, 2003). To date, only three peroxins with a potential role in PMP targeting have been identified, namely Pex3p (Hettema et al., 2000;South et al., 2000), Pex16p in mammals (South and Gould, 1999;Honsho et al., 2002), and Pex19p (Gö tte et al., 1998;Matsuzono et al., 1999;Snyder et al., 1999;Soukupova et al., 1999). In cells lacking any of these proteins, PMPs are either degraded or mistargeted to other subcellular compartments such as mitochondria, the endoplasmic reticulum (ER) and membranes of unknown origin (Ghaedi et al., 2000;Hettema et al., 2000;Sacksteder et al., 2000).In accordance with a distinct pathway, PMPs use neither PTS1 nor PTS2. The targeting signals of PMPs (mPTS) that direct and insert PMPs into the peroxisomal membrane have been determined for a number of PMPs of several species. Despite some differences, a picture emerged from these studies of a targeting signal consisting of one or more transmembrane domains in conjunction with a short sequence, which contains either a cluster of basic residues or a mixture of basic and hydrophobic amino acids (Dyer et al., 1996;Baerends et al., 2000b;Pause et al., 2000;...

Lassa virus (LASV) causes a deadly haemorrhagic fever in humans, killing several thousand people in West Africa annually. For 40 years, the Natal multimammate rat, Mastomys natalensis, has been assumed to be the sole host of LASV. We found evidence that LASV is also hosted by other rodent species: the African wood mouse Hylomyscus pamfi in Nigeria, and the Guinea multimammate mouse Mastomys erythroleucus in both Nigeria and Guinea. Virus strains from these animals were isolated in the BSL-4 laboratory and fully sequenced. Phylogenetic analyses of viral genes coding for glycoprotein, nucleoprotein, polymerase and matrix protein show that Lassa strains detected in M. erythroleucus belong to lineages III and IV. The strain from H. pamfi clusters close to lineage I (for S gene) and between II & III (for L gene). Discovery of new rodent hosts has implications for LASV evolution and its spread into new areas within West Africa.

Tail-anchored proteins contain a single transmembrane domain (TMD) followed by a short C-terminal domain extending into the organellar lumen. Tail-anchored proteins are thought to target to the correct subcellular compartment by virtue of general physicochemical properties of their C-termini; however, the machineries that enable correct sorting remain largely elusive. Here we analyzed targeting of the human peroxisomal tail-anchored protein PEX26. Its C-terminal-targeting signal contains two binding sites for PEX19, the import receptor for several peroxisomal membrane proteins. One PEX19-binding site overlapped with the TMD, the other was contained within the luminal domain. Although the PEX19-binding site containing the TMD targeted to peroxisomes to some extent, the luminal site proved essential for correct targeting of the full-length protein, as it prevented PEX26 from mislocalization to mitochondria. Its function as a targeting motif was proved by its ability to insert a heterologous TMD-containing fragment into the peroxisomal membrane. Finally we show that PEX19 is essential for PEX26 import. Analysis of the yeast tail-anchored protein Pex15p revealed that it also harbors a luminal PEX19-binding site that acts as a peroxisomal-targeting motif. We conclude that C-terminal PEX19-binding sites mark tail-anchored proteins for delivery to peroxisomes.

Rift Valley fever virus (RVFV; Phlebovirus, Bunyaviridae) is an emerging zoonotic mosquito-borne pathogen of high relevance for human and animal health. Successful strategies of intervention in RVFV transmission by its mosquito vectors and the prevention of human and veterinary disease rely on a better understanding of the mechanisms that govern RVFV-vector interactions. Despite its medical importance, little is known about the factors that govern RVFV replication, dissemination, and transmission in the invertebrate host. Here we studied the role of the antiviral RNA interference immune pathways in the defense against RVFV in natural vector mosquitoes and mosquito cells and draw comparisons to the model insect Drosophila melanogaster. We found that RVFV infection induces both the exogenous small interfering RNA (siRNA) and piRNA pathways, which contribute to the control of viral replication in insects. Furthermore, we demonstrate the production of virus-derived piRNAs in Culex quinquefasciatus mosquitoes. Understanding these pathways and the targets within them offers the potential of the development of novel RVFV control measures in vector-based strategies.