Efficient Rescue of Recombinant Lassa Virus Reveals the Influence of S Segment Noncoding Regions on Virus Replication and Virulence

Albariño, César G.; Bird, Brian H.; Chakrabarti, Ayan K.; Dodd, Kimberly A.; Erickson, Bobbie R.; Nichol, Stuart T.

doi:10.1128/jvi.02556-10

Cited by 46 publications

(43 citation statements)

References 26 publications

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“…To obtain a recombinant Lujo virus, we focused our initial efforts on sequencing the genome termini, as these are essential cis elements of viral promoters (6,15,17,30). The sequences of the genomic RNA termini of Pichínde virus (33), Tacaríbe virus (34), and LCMV (14) implied the presence of a nontemplated pppG originating from internal priming by a 5= dinucleotide triphosphate (5= pppGpC) and realigning the primer with the terminal 3= G. In contrast to other arenavirus results, we detected not only a possible nontemplated 5= pppG in Lujo virus but also an additional 3= C at the termini of both segments, albeit a significant proportion of S termini end with 3= G, as in other arenaviruses (1,2,5,14,27,34). Our data suggest that Lujo virus genome termini differ significantly from those of other arenaviruses.…”

Section: Discussioncontrasting

confidence: 53%

“…The L segment reporter plasmid was obtained by replacing the Z protein ORF of pLJL IGR-104 with Gaussia luciferase (pLJL GLuc-IGR 104). The mutation of SDD 1353 to SAA was introduced into pLJL-GLuc IGR-104 to inactivate L-RdRp (pLJL-Gluc SDD) (1,2,7,19,35). pLJL IGR-140 and pLJL-GLuc IGR-140 were obtained by replacing the IGR of pLJL IGR-104 or pLJL-GLuc IGR-104 with L-IGR-140 from a clone comprising the new consensus.…”

Section: Methodsmentioning

confidence: 99%

“…Viral RNA from this isolate was therefore used as the template to generate all the Lujo virus cDNA clones. Full-length Lujo virus antigenomic segments were cloned into the same modified V(0.0) T7 expression vector used to rescue Lassa virus from cDNA (2). [The detailed cloning strategy of the L and S segments and the modified V(0.0) vector sequence are available upon request.]…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Reverse Genetics Recovery of Lujo Virus and Role of Virus RNA Secondary Structures in Efficient Virus Growth

Bergeron

Chakrabarti

Bird

et al. 2012

J Virol

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Arenaviruses are rodent-borne viruses with a bisegmented RNA genome. A genetically unique arenavirus, Lujo virus, was recently discovered as the causal agent of a nosocomial outbreak of acute febrile illness with hemorrhagic manifestations in Zambia and South Africa. The outbreak had a case fatality rate of 80%. A reverse genetics system to rescue infectious Lujo virus from cDNA was established to investigate the biological properties of this virus. Sequencing the genomic termini showed unique nucleotides at the 3′ terminus of the S segment promoter element. While developing this system, we discovered that reconstructing infectious Lujo virus using the previously reported L segment intergenic region (IGR), comprising the arenaviral transcription termination signal, yielded an attenuated Lujo virus. Resequencing revealed that the correct L segment IGR was 36 nucleotides longer, and incorporating it into the reconstructed Lujo virus restored the growth rate to that of the authentic clinical virus isolate. These additional nucleotides were predicted to more than double the free energy of the IGR main stem-loop structure. In addition, incorporating the newly determined L-IGR into a replicon reporter system enhanced the expression of a luciferase reporter L segment. Overall, these results imply that an extremely stable secondary structure within the L-IGR is critical for Lujo virus propagation and viral protein production. The technology for producing recombinant Lujo virus now provides a method to precisely investigate the molecular determinants of virulence of this newly identified pathogen.

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Section: Discussioncontrasting

confidence: 53%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Reverse Genetics Recovery of Lujo Virus and Role of Virus RNA Secondary Structures in Efficient Virus Growth

Bergeron

Chakrabarti

Bird

et al. 2012

J Virol

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“…The Lassa virus Josiah strain complete sequence was downloaded from the National Center for Biotechnology Information (Accessions HQ688674 and HQ688672). 70 Antigen sequences were downloaded in GenPept format, where the accession number and corresponding amino acid sequence of each of the 4 antigens were exported and then uploaded to an inhouse database. Per antigen, the amino acid sequence was parsed into 9-mer peptides overlapping by 8 amino acids and analyzed using EpiMatrix 1.2 to identify potential T cell epitopes.…”

Section: Immunogenic Epitopes Identificationmentioning

confidence: 99%

VaxCelerate II: Rapid development of a self-assembling vaccine for Lassa fever

Leblanc

Moise

Luza

et al. 2014

Human Vaccines & Immunotherapeutics

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Development of effective vaccines against emerging infectious diseases (EID) can take as much or more than a decade to progress from pathogen isolation/identification to clinical approval. As a result, conventional approaches fail to produce field-ready vaccines before the EID has spread extensively. Lassa is a prototypical emerging infectious disease endemic to West Africa for which no successful vaccine is available. We established the VaxCelerate Consortium to address the need for more rapid vaccine development by creating a platform capable of generating and pre-clinically testing a new vaccine against specific pathogen targets in less than 120 d. A self-assembling vaccine is at the core of the approach. It consists of a fusion protein composed of the immunostimulatory Mycobacterium tuberculosis heat shock protein 70 (MtbHSP70) and the biotin binding protein, avidin. Mixing the resulting protein (MAV) with biotinylated pathogen-specific immunogenic peptides yields a self-assembled vaccine (SAV). To meet the time constraint imposed on this project, we used a distributed R&D model involving experts in the fields of protein engineering and production, bioinformatics, peptide synthesis/design and GMP/GLP manufacturing and testing standards. SAV immunogenicity was first tested using H1N1 influenza specific peptides and the entire VaxCelerate process was then tested in a mock live-fire exercise targeting Lassa fever virus. We demonstrated that the Lassa fever vaccine induced significantly increased class II peptide specific interferon-g CD4C T cell responses in HLA-DR3 transgenic mice compared to peptide or MAV alone controls. We thereby demonstrated that our SAV in combination with a distributed development model may facilitate accelerated regulatory review by using an identical design for each vaccine and by applying safety and efficacy assessment tools that are more relevant to human vaccine responses than current animal models.

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“…The recent development of reverse genetics systems for JUNV [43, 44] and LASV [45, 46] would facilitate the elucidation of the genetic determinants of JUNV and LASV virulence. This, in turn, should help with the design of safer live-attenuated arenavirus vaccines by minimizing concerns related to reversion of virulence, establishment of persistent infection, and in general conditions associated with high risk for replicating viruses including immunocompromised individuals.…”

Section: Introductionmentioning

confidence: 99%

Reverse Genetics Approaches to Control Arenavirus

Martínez-Sobrido¹,

Cheng²,

Torre³

2016

Vaccine Design

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Several arenavirus cause hemorrhagic fever disease in humans and pose a significant public health problem in their endemic regions. To date, no licensed vaccines are available to combat human arenavirus infections, and anti-arenaviral drug therapy is limited to an off-label use of ribavirin that is only partially effective. The development of arenavirus reverse genetics approaches provides investigators with a novel and powerful approach for the investigation of the arenavirus molecular and cell biology. The use of cell-based minigenome systems has allowed examining the cis- and trans-acting factors involved in arenavirus replication and transcription and the identification of novel anti-arenaviral drug targets without requiring the use of live forms of arenaviruses. Likewise, it is now feasible to rescue infectious arenaviruses entirely from cloned cDNAs containing predetermined mutations in their genomes to investigate virus-host interactions and mechanisms of pathogenesis, as well as to facilitate screens to identify anti-arenaviral drugs and development of novel live-attenuated arenavirus vaccines. Recently, reverse genetics have also allowed the generation of tri-segmented arenaviruses expressing foreign genes, facilitating virus detection and opening the possibility of implementing live-attenuated arenavirus-based vaccine vector approaches. Likewise, the development of single-cycle infectious, reporter-expressing, arenaviruses has provided a new experimental method to study some aspects of the biology of highly pathogenic arenaviruses without the requirement of high-security biocontainment required to study HF-causing arenaviruses. In this chapter we summarize the current knowledge on arenavirus reverse genetics and the implementation of plasmid-based reverse genetics techniques for the development of arenavirus vaccines and vaccine vectors.

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Efficient Rescue of Recombinant Lassa Virus Reveals the Influence of S Segment Noncoding Regions on Virus Replication and Virulence

Cited by 46 publications

References 26 publications

Reverse Genetics Recovery of Lujo Virus and Role of Virus RNA Secondary Structures in Efficient Virus Growth

Reverse Genetics Recovery of Lujo Virus and Role of Virus RNA Secondary Structures in Efficient Virus Growth

VaxCelerate II: Rapid development of a self-assembling vaccine for Lassa fever

Reverse Genetics Approaches to Control Arenavirus

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