Beate Becker-Ziaja

Replicon System for Lassa Virus

ABSTRACT Lassa virus is endemic to West Africa and causes hemorrhagic fever in humans. To facilitate the functional analysis of this virus, a replicon system was developed based on Lassa virus strain AV. Genomic and antigenomic minigenomes (MG) were constructed consisting of the intergenic region of S RNA and a reporter gene (Renilla luciferase) in antisense orientation, flanked by the 5′ and 3′ untranslated regions of S RNA. MGs were expressed under the control of the T7 promoter. Nucleoprotein (NP), L protein, and Z protein were expressed from plasmids containing the T7 promoter and internal ribosomal entry site. Transfection of cells stably expressing T7 RNA polymerase (BSR T7/5) with MG in the form of DNA or RNA and plasmids for the expression of NP and L protein resulted in high levels of Renilla luciferase expression. The replicon system was optimized with respect to the ratio of the transfected constructs and by modifying the 5′ end of the MG. Maximum activity was observed 24 to 36 h after transfection with a signal-to-noise ratio of 2 to 3 log units. Northern blot analysis provided evidence for replication and transcription of the MG. Z protein downregulated replicon activity close to background levels. Treatment with ribavirin and alpha interferon inhibited replicon activity, suggesting that both act on the level of RNA replication, transcription, or ribonucleoprotein assembly. In conclusion, this study describes the first replicon system for a highly pathogenic arenavirus. It is a tool for investigating the mechanisms of replication and transcription of Lassa virus and may facilitate the testing of antivirals outside a biosafety level 4 laboratory.

Unique human immune signature of Ebola virus disease in Guinea

Despite the magnitude of the Ebola virus disease (EVD) outbreak in West Africa, there is still a fundamental lack of knowledge about the pathophysiology of EVD. In particular, very little is known about human immune responses to Ebola virus. Here we evaluate the physiology of the human T cell immune response in EVD patients at the time of admission to the Ebola Treatment Center in Guinea, and longitudinally until discharge or death. Through the use of multiparametric flow cytometry established by the European Mobile Laboratory in the field, we identify an immune signature that is unique in EVD fatalities. Fatal EVD was characterized by a high percentage of CD4+ and CD8+ T cells expressing the inhibitory molecules CTLA-4 and PD-1, which correlated with elevated inflammatory markers and high virus load. Conversely, surviving individuals showed significantly lower expression of CTLA-4 and PD-1 as well as lower inflammation, despite comparable overall T cell activation. Concomitant with virus clearance, survivors mounted a robust Ebola-virus-specific T cell response. Our findings suggest that dysregulation of the T cell response is a key component of EVD pathophysiology.

Molecular Diagnostics for Lassa Fever at Irrua Specialist Teaching Hospital, Nigeria: Lessons Learnt from Two Years of Laboratory Operation

Background Lassa fever is a viral hemorrhagic fever endemic in West Africa. However, none of the hospitals in the endemic areas of Nigeria has the capacity to perform Lassa virus diagnostics. Case identification and management solely relies on non-specific clinical criteria. The Irrua Specialist Teaching Hospital (ISTH) in the central senatorial district of Edo State struggled with this challenge for many years. Methodology/Principal Findings A laboratory for molecular diagnosis of Lassa fever, complying with basic standards of diagnostic PCR facilities, was established at ISTH in 2008. During 2009 through 2010, samples of 1,650 suspected cases were processed, of which 198 (12%) tested positive by Lassa virus RT-PCR. No remarkable demographic differences were observed between PCR-positive and negative patients. The case fatality rate for Lassa fever was 31%. Nearly two thirds of confirmed cases attended the emergency departments of ISTH. The time window for therapeutic intervention was extremely short, as 50% of the fatal cases died within 2 days of hospitalization—often before ribavirin treatment could be commenced. Fatal Lassa fever cases were older (p = 0.005), had lower body temperature (p<0.0001), and had higher creatinine (p<0.0001) and blood urea levels (p<0.0001) than survivors. Lassa fever incidence in the hospital followed a seasonal pattern with a peak between November and March. Lassa virus sequences obtained from the patients originating from Edo State formed—within lineage II—a separate clade that could be further subdivided into three clusters. Conclusions/Significance Lassa fever case management was improved at a tertiary health institution in Nigeria through establishment of a laboratory for routine diagnostics of Lassa virus. Data collected in two years of operation demonstrate that Lassa fever is a serious public health problem in Edo State and reveal new insights into the disease in hospitalized patients.

Application of real-time PCR for testing antiviral compounds against Lassa virus, SARS coronavirus and Ebola virus in vitro

Abstract This report describes the application of real-time PCR for testing antivirals against highly pathogenic viruses such as Lassa virus, SARS coronavirus and Ebola virus. The test combines classical cell culture with a quantitative real-time PCR read-out. The assay for Lassa virus was validated with ribavirin, which showed an IC50 of 9μg/ml. Small-scale screening identified a class of imidazole nucleoside/nucleotide analogues with antiviral activity against Lassa virus. The analogues contained either dinitrile or diester groups at the imidazole 4,5-positions, and many of which possessed an acyclic sugar or sugar phosphonate moiety at the imidazole 1-position. The IC50 values of the most active compounds ranged from 5 to 21μg/ml. The compounds also inhibited replication of SARS coronavirus and Ebola virus in analogous assays, although to a lesser extent than Lassa virus.

Mutational Analysis of the Lassa Virus Promoter

ABSTRACT The promoter sequences directing viral gene expression and genome replication of arenaviruses reside within the 3′ and 5′ termini of each RNA segment. The terminal 19 nucleotides at both ends are highly conserved among all arenavirus species and are almost completely complementary to each other. This study aimed at characterizing the Lassa virus promoter in detail. The relevance of each position in the promoter was studied by site-directed mutagenesis using the Lassa virus minireplicon system. The data indicate that the Lassa virus promoter functions as a duplex, regulates transcription and replication in a coordinated manner, and is composed of two functional elements, a sequence-specific region from residue 1 to 12 and a variable complementary region from residue 13 to 19. The first region appears to interact with the replication complex mainly via base-specific interactions, while in the second region solely base pairing between 3′ and 5′ promoter ends is important for promoter function.

Current Molecular Epidemiology of Lassa Virus in Nigeria

ABSTRACT 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.

Mopeia virus-related arenavirus in natal multimammate mice, Morogoro, Tanzania

A serosurvey involving 2,520 small mammals from Tanzania identified a hot spot of arenavirus circulation in Morogoro. Molecular screening detected a new arenavirus in Natal multimammate mice (Mastomys natalensis), Morogoro virus, related to Mopeia virus. Only a small percentage of mice carry Morogoro virus, although a large proportion shows specific antibodies.

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

ABSTRACT 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.

Domain Structure of Lassa Virus L Protein

ABSTRACT 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.

Lassa Fever, Nigeria, 2005–2008

To the Editor: Lassa fever affects ≈100,000 persons per year in West Africa (1). The disease is caused by Lassa virus, an arenavirus, and is associated with bleeding and organ failure. The case-fatality rate in hospitalized patients is 10%–20%. The reservoir of the virus is multimammate mice (Mastomys natalensis). Investigations in the 1970s and 1980s pointed to the existence of 3 disease-endemic zones within Nigeria: the northeastern region around Lassa, the central region around Jos, and the southern region around Onitsha (2,3). The current epidemiologic situation is less clear because no surveillance system is in place. In 2003 and 2004, we conducted a hospital-based survey in Irrua, which demonstrated ongoing transmission of the virus in Edo State, Nigeria (4). Since then, laboratory capacity at the University of Lagos for diagnosing Lassa fever has been improved and used for small-scale passive surveillance in other parts of the country. Public health officials or hospital staff reported suspected cases. Blood samples were sent to Lagos, or staff from Lagos collected samples on site. Confirmatory testing, sequencing, and virus isolation were performed at the Bernhard Nocht Institute for Tropical Medicine in Hamburg, Germany. Primary testing was done by reverse transcription–PCR (RT-PCR) that targeted the glycoprotein (GP) gene (5,6). An RT-PCR that targeted the large (L) gene was used as a secondary test (7), and PCR products were sequenced. Serologic testing for Lassa virus–specific immunoglobulin (Ig) G and IgM was performed by immunofluorescent antibody test using Vero cells infected with Lassa virus. Virus isolation with Vero cells was conducted in the BioSafety Level 4 laboratory in Hamburg. From 2005 through 2008, 10 cases of Lassa fever were confirmed by virus detection (cases 3–10) or implicated by epidemiologic investigation and serologic testing (cases 1 and 2) (Appendix Table). Case-patients 1–4 were involved in a nosocomial outbreak that occurred in February 2005 at the Ebonyi State University Teaching Hospital (EBSUTH) in Abakaliki. Retrospective investigation suggests the following transmission chain. The presumed index case-patient was a male nurse living in Onitsha, who became ill on January 21, 2005, and traveled ≈200 km to EBSUTH for better medical treatment. The detection of Lassa virus–specific IgM during his convalescent phase indicates that he had Lassa fever. The second case-patient was a female nurse who had contact with the index case-patient on February 4. She was admitted on February 7 and died 6 days later. Her clinical features were compatible with Lassa fever, but laboratory confirmation is lacking because specimens were not collected. Two additional case-patients among hospital staff (case-patients 3 and 4) were seen on February 21; each had had contact with case-patient 2. Case-patient 3 took care of case-patient 2 and slept in the same room with her for 4 days. Lassa fever was confirmed in case-patients 3 and 4 by RT-PCR as well as by IgM and IgG seroconversion in the surviving patient (case-patient 3). Case-patient 4, a pregnant nurse, had a spontaneous abortion and died on day 9 of hospitalization. Sequencing the GP and L gene PCR fragments showed that case-patients 3 and 4 were infected with the same virus strain (100% identity). In March and April 2005, blood was collected from 50 hospital staff members (including those who had had contact with the case-patients) and screened for Lassa virus–specific IgM and IgG. No positive blood samples were found, which indicated that no additional staff members were involved in the outbreak. Case-patients 5 and 6 were admitted to EBSUTH in 2008 on January 17 and March 5, respectively. Both were medical doctors, one at a local hospital and the other at EBSUTH, and both died. Encephalopathy with generalized seizures and loss of consciousness preceded death in both cases. The source of infection is unknown, although it is likely that they became infected while they treated patients without knowing they had Lassa fever. In agreement with the epidemiology, the viruses from the 2 patients were similar, though not identical (89% and 87% identity in the GP and L genes, respectively). Cases 7 to 10 occurred in Abuja and Jos from December 2007 through March 2008. Healthcare workers appeared not to be involved, and no molecular epidemiologic evidence indicated that transmission occurred among the 3 case-patients from Jos (94–97% and 90–94% identity in the GP and L genes, respectively). In conjunction with our previous report (4), the cases presented here demonstrate current Lassa fever activity in the states of Edo, Ebonyi, Federal Capital Territory, and Plateau. These findings correspond to early reports on Lassa fever in southern and central parts of Nigeria. That healthcare workers are still at as high a risk of contracting and dying from the disease as they were 20 years ago (8) is alarming. A key to solving this problem would be the establishment of diagnostic facilities that can provide rapid molecular testing at referral centers in the disease-endemic zones. This testing would facilitate appropriate case and contact management, including early treatment and postexposure prophylaxis with ribavirin, and eventually raise awareness that Lassa fever should be considered in every severe febrile illness in these regions.