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1.  Hospital-Based Surveillance for Viral Hemorrhagic Fevers and Hepatitides in Ghana 
Background
Viral hemorrhagic fevers (VHF) are acute diseases associated with bleeding, organ failure, and shock. VHF may hardly be distinguished clinically from other diseases in the African hospital, including viral hepatitis. This study was conducted to determine if VHF and viral hepatitis contribute to hospital morbidity in the Central and Northern parts of Ghana.
Methodology/Principal Findings
From 2009 to 2011, blood samples of 258 patients with VHF symptoms were collected at 18 hospitals in Ashanti, Brong-Ahafo, Northern, Upper West, and Upper East regions. Patients were tested by PCR for Lassa, Rift Valley, Crimean-Congo, Ebola/Marburg, and yellow fever viruses; hepatitis A (HAV), B (HBV), C (HCV), and E (HEV) viruses; and by ELISA for serological hepatitis markers. None of the patients tested positive for VHF. However, 21 (8.1%) showed anti-HBc IgM plus HBV DNA and/or HBsAg; 37 (14%) showed HBsAg and HBV DNA without anti-HBc IgM; 26 (10%) showed anti-HAV IgM and/or HAV RNA; and 20 (7.8%) were HCV RNA-positive. None was positive for HEV RNA or anti-HEV IgM plus IgG. Viral genotypes were determined as HAV-IB, HBV-A and E, and HCV-1, 2, and 4.
Conclusions/Significance
VHFs do not cause significant hospital morbidity in the study area. However, the incidence of acute hepatitis A and B, and hepatitis B and C with active virus replication is high. These infections may mimic VHF and need to be considered if VHF is suspected. The data may help decision makers to allocate resources and focus surveillance systems on the diseases of relevance in Ghana.
Author Summary
Ghana is endemic for yellow fever and lies between two Lassa fever endemic areas — Guinea, Liberia, Sierra Leone, and Mali in the West, and Nigeria in the East. Ebola hemorrhagic fever has been documented in the neighboring Cote d'Ivoire. Thus, it is plausible that the latter VHFs also occur in Ghana, and there have been rumors of cases, which present like VHF in the north of the country. Our study aimed at verifying or disproving this suspicion. At 18 hospital-based study sites in the Central and Northern part of the country, samples from 258 patients with VHF symptoms were collected and tested for various VHF by PCR. As viral hepatitis is an important differential diagnosis of yellow fever, we also tested for several serological and molecular hepatitis markers. Rather surprisingly, VHFs were not detected, indicating that, even if they are endemic in the North of Ghana, they do not significantly contribute to hospital morbidity. However, a large fraction of patients showed markers of acute hepatitis A, and active hepatitis B and C. Children were mainly affected by hepatitis A, while adults were affected by hepatitis B and C. Hepatitis A and B are vaccine-preventable, and chronic hepatitis B and C are treatable diseases. Further efforts are needed to reduce the burden of these diseases in Ghana.
doi:10.1371/journal.pntd.0002435
PMCID: PMC3777898  PMID: 24069490
2.  Current Molecular Epidemiology of Lassa Virus in Nigeria▿§ 
Journal of Clinical Microbiology  2011;49(3):1157-1161.
Recent Lassa virus strains from Nigeria were completely or partially sequenced. Phylogenetic analysis revealed the predominance of lineage II and III strains, the existence of a previously undescribed (sub)lineage in Nigeria, and the directional spread of virus in the southern part of the country. The Bayesian analysis also provided estimates for divergence times within the Lassa virus clade.
doi:10.1128/JCM.01891-10
PMCID: PMC3067713  PMID: 21191050
3.  Domain Structure of Lassa Virus L Protein ▿  
Journal of Virology  2010;85(1):324-333.
The 200-kDa L protein of arenaviruses plays a central role in viral genome replication and transcription. This study aimed at providing evidence for the domain structure of L protein by combining bioinformatics with a stepwise mutagenesis approach using the Lassa virus minireplicon system. Potential interdomain linkers were predicted using various algorithms. The prediction was challenged by insertion of flexible sequences into the predicted linkers. Insertion of 5 or 10 amino acid residues was tolerated at seven sites (S407, G446, G467, G774, G939, S1952, and V2074 in Lassa virus AV). At two of these sites, G467 and G939, L protein could be split into an N-terminal and a C-terminal part, which were able to trans-complement each other and reconstitute a functional complex upon coexpression. Coimmunoprecipitation studies revealed physical interaction between the N- and C-terminal domains, irrespective of whether L protein was split at G467 or G939. In confocal immunofluorescence microscopy, the N-terminal domains showed a dot-like, sometimes perinuclear, cytoplasmic distribution similar to that of full-length L protein, while the C-terminal domains were homogenously distributed in cytoplasm. The latter were redistributed into the dot-like structures upon coexpression with the corresponding N-terminal domain. In conclusion, this study demonstrates two interdomain linkers in Lassa virus L protein, at G467 and G939, suggesting that L protein is composed of at least three structural domains spanning residues 1 to 467, 467 to 939, and 939 to 2220. The first domain seems to mediate accumulation of L protein into cytoplasmic dot-like structures.
doi:10.1128/JVI.00721-10
PMCID: PMC3014181  PMID: 20980514
4.  Improved Detection of Lassa Virus by Reverse Transcription-PCR Targeting the 5′ Region of S RNA▿  
Journal of Clinical Microbiology  2010;48(6):2009-2013.
The method of choice for the detection of Lassa virus is reverse transcription (RT)-PCR. However, the high degree of genetic variability of the virus poses a problem with the design of RT-PCR assays that will reliably detect all strains. Recently, we encountered difficulties in detecting some strains from Liberia and Nigeria in a commonly used glycoprotein precursor (GPC) gene-specific RT-PCR assay (A. H. Demby, J. Chamberlain, D. W. Brown, and C. S. Clegg, J. Clin. Microbiol. 32:2898-2903, 1994), which prompted us to revise the protocol. The design of the new assay, the GPC RT-PCR/2007 assay, took into account 62 S RNA sequences from all countries where Lassa fever is endemic, including 40 sequences generated from the strains in our collection. The analytical sensitivity of the new assay was determined with 11 strains from Sierra Leone, Liberia, Ivory Coast, and Nigeria by probit analysis; the viral loads detectable with a probability of 95% ranged from 342 to 2,560 S RNA copies/ml serum, which corresponds to 4 to 30 S RNA copies/assay. The GPC RT-PCR/2007 assay was validated with 77 serum samples and 1 cerebrospinal fluid sample from patients with laboratory-confirmed Lassa fever. The samples mainly originated from Liberia and Nigeria and included strains difficult to detect in the assay of 1994. The GPC RT-PCR/2007 assay detected virus in all clinical specimens (100% sensitivity). In conclusion, a new RT-PCR assay, based in part on the protocol developed by Demby et al. in 1994, for the detection of Lassa virus is described. Compared to the assay developed in 1994, the GPC RT-PCR/2007 assay offers improved sensitivity for the detection of Liberian and Nigerian Lassa virus strains.
doi:10.1128/JCM.02351-09
PMCID: PMC2884523  PMID: 20351210
5.  The N-Terminal Domain of the Arenavirus L Protein Is an RNA Endonuclease Essential in mRNA Transcription 
PLoS Pathogens  2010;6(9):e1001038.
Arenaviridae synthesize viral mRNAs using short capped primers presumably acquired from cellular transcripts by a ‘cap-snatching’ mechanism. Here, we report the crystal structure and functional characterization of the N-terminal 196 residues (NL1) of the L protein from the prototypic arenavirus: lymphocytic choriomeningitis virus. The NL1 domain is able to bind and cleave RNA. The 2.13 Å resolution crystal structure of NL1 reveals a type II endonuclease α/β architecture similar to the N-terminal end of the influenza virus PA protein. Superimposition of both structures, mutagenesis and reverse genetics studies reveal a unique spatial arrangement of key active site residues related to the PD…(D/E)XK type II endonuclease signature sequence. We show that this endonuclease domain is conserved and active across the virus families Arenaviridae, Bunyaviridae and Orthomyxoviridae and propose that the arenavirus NL1 domain is the Arenaviridae cap-snatching endonuclease.
Author Summary
The Arenaviridae virus family includes several life-threatening human pathogens that cause meningitis or hemorrhagic fever. These RNA viruses replicate and transcribe their genome using an RNA synthesis machinery for which no structural data currently exist. They synthesize viral mRNAs using short capped primers presumably acquired from cellular transcripts by a ‘cap-snatching’ mechanism thought to involve the large L protein, which carries RNA-dependent RNA polymerase signature sequences. Here, we report the crystal structure and functional characterization of an isolated N-terminal domain of the L protein (NL1) from the prototypic arenavirus: lymphocytic choriomeningitis virus. The NL1 domain is able to bind and cleave RNA. The 2.13 Å resolution crystal structure of NL1 reveals a type II endonuclease α/β architecture similar to the N-terminal end of the influenza virus PA protein. Superimposition of both structures and mutagenesis studies reveal a unique spatial arrangement of key active site residues related to the PD…(D/E)XK type II endonuclease signature sequence. Reverse genetic studies show that mutation of active site residues selectively abolish transcription, not replication. We show that this endonuclease domain is conserved and active across the virus families: Arenaviridae, Bunyaviridae and Orthomyxoviridae and propose that the arenavirus NL1 domain is the Arenaviridae cap-snatching endonuclease.
doi:10.1371/journal.ppat.1001038
PMCID: PMC2940758  PMID: 20862324
6.  An N-Terminal Region of Lassa Virus L Protein Plays a Critical Role in Transcription but Not Replication of the Virus Genome ▿ †  
Journal of Virology  2009;84(4):1934-1944.
The central domain of the 200-kDa Lassa virus L protein is a putative RNA-dependent RNA polymerase. N- and C-terminal domains may harbor enzymatic functions important for viral mRNA synthesis, including capping enzymes or cap-snatching endoribonucleases. In the present study, we have employed a large-scale mutagenesis approach to map functionally relevant residues in these regions. The main targets were acidic (Asp and Glu) and basic residues (Lys and Arg) known to form catalytic and binding sites of capping enzymes and endoribonucleases. A total of 149 different mutants were generated and tested in the Lassa virus replicon system. Nearly 25% of evolutionarily highly conserved acidic and basic side chains were dispensable for function of L protein in the replicon context. The vast majority of the remaining mutants had defects in both transcription and replication. Seven residues (Asp-89, Glu-102, Asp-119, Lys-122, Asp-129, Glu-180, and Arg-185) were selectively important for mRNA synthesis. The phenotype was particularly pronounced for Asp-89, Glu-102, and Asp-129, which were indispensable for transcription but could be replaced by a variety of amino acid residues without affecting genome replication. Bioinformatics disclosed the remote similarity of this region to type IIs endonucleases. The mutagenesis was complemented by experiments with the RNA polymerase II inhibitor α-amanitin, demonstrating dependence of viral transcription from the cellular mRNA pool. In conclusion, this paper describes an N-terminal region in L protein being important for mRNA, but not genome synthesis. Bioinformatics and cell biological experiments lend support to the hypothesis that this region could be part of a cap-snatching enzyme.
doi:10.1128/JVI.01657-09
PMCID: PMC2812395  PMID: 20007273
7.  Mutational Evidence for a Structural Model of the Lassa Virus RNA Polymerase Domain and Identification of Two Residues, Gly1394 and Asp1395, That Are Critical for Transcription but Not Replication of the Genome▿  
Journal of Virology  2008;82(20):10207-10217.
The RNA-dependent RNA polymerase (RdRp) of arenaviruses is an integral part of the L protein, a 200-kDa multifunctional and multidomain protein. In view of the paucity of structural data, we recently proposed a model for the RdRp domain of arenaviruses based on the folding of RdRps of plus-strand viruses (S. Vieth et al., Virology 318:153-168, 2004). In the present study, we have chosen a large-scale mutagenesis approach to gain insight into the structure and function of the Lassa virus RdRp domain. A total of 180 different mutants of the domain were generated by using a novel PCR-based mutagenesis technique and tested in the context of the Lassa virus replicon system. Nearly all residues, which were essential for function, clustered in the center of the three-dimensional model including the catalytic site, while residues that were less important for function mapped to the periphery of the model. The combined bioinformatics and mutagenesis data allowed deducing candidate residues for ligand interaction. Mutation of two adjacent residues in the putative palm-thumb subdomain junction, G1394 and D1395 (strain AV), led to a defect in mRNA synthesis but did not affect antigenomic RNA synthesis. In conclusion, the data provide circumstantial evidence for the existence of an RdRp domain between residues 1040 and 1540 of the Lassa virus L protein and the folding model of the domain. A functional element within the RdRp was identified, which is important for transcription but not replication of the genome.
doi:10.1128/JVI.00220-08
PMCID: PMC2566270  PMID: 18667512

Results 1-7 (7)