Reverse genetics recovery of Lujo virus and role of virus RNA secondary structures in efficient virus growth.
ABSTRACT: 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.
Project description:To identify host factors associated with arenavirus virulence, we used a cynomolgus macaque model to evaluate the pathogenesis of Lujo virus (LUJV), a recently emerged arenavirus that caused an outbreak of severe viral hemorrhagic fever in southern Africa. In contrast to human cases, LUJV caused mild, nonlethal illness in macaques. We then compared this to contrasting clinical outcomes during arenavirus infection, specifically to samples obtained from macaques infected with three highly pathogenic lines of Lassa virus (LASV), the causative agent of Lassa fever (LF). We assessed gene expression in peripheral blood mononuclear cells (PBMC) and determined genes that significantly changed expression relative to that in uninfected animals over the course of infection. We detected a 72-h delay in the induction of host responses to infection during LUJV infection compared to that of the animals infected with LASV. This included genes associated with inflammatory and antiviral responses and was particularly apparent among groups of genes promoting cell death. We also observed early differential expression of a subset of genes specific to LUJV infection that accounts for the delayed inflammatory response. Cell type enrichment analysis suggested that host response induction delay and an LUJV-specific profile are due to a different proportion of natural killer cells responding in LUJV infection than that in the LASV-infected animals. Together, these data indicate that delayed proinflammatory and proapoptotic host responses to arenavirus infection could ameliorate disease severity. This conclusion provides insight into the cellular and molecular mechanisms of arenaviral hemorrhagic fever and suggests potential strategies for therapeutic development.Old World arenaviruses are significant human pathogens that often are associated with high mortality. However, mechanisms underlying disease severity and virulence in arenavirus hemorrhagic fever are largely unknown, particularly regarding host responses that contribute to pathogenicity. This study describes a comparison between Lujo and Lassa virus infection in cynomolgus macaques. Lujo virus-infected macaques developed only mild illness, while Lassa virus-infected macaques developed severe illness consistent with Lassa fever. We determined that mild disease is associated with a delay in host expression of genes linked to virulence, such as those causing inflammation and cell death, and with distinct cell types that may mediate this delay. This is the first study to associate the timing and directionality of gene expression with arenaviral pathogenicity and disease outcome and evokes new potential approaches for developing effective therapeutics for treating these deadly emerging pathogens.
Project description:In 2008 a nosocomial outbreak of five cases of viral hemorrhagic fever due to a novel arenavirus, Lujo virus, occurred in Johannesburg, South Africa. Lujo virus is only the second pathogenic arenavirus, after Lassa virus, to be recognized in Africa and the first in over 40 years. Because of the remote, resource-poor, and often politically unstable regions where Lassa fever and other viral hemorrhagic fevers typically occur, there have been few opportunities to undertake in-depth study of their clinical manifestations, transmission dynamics, pathogenesis, or response to treatment options typically available in industrialized countries.We describe the clinical features of five cases of Lujo hemorrhagic fever and summarize their clinical management, as well as providing additional epidemiologic detail regarding the 2008 outbreak. Illness typically began with the abrupt onset of fever, malaise, headache, and myalgias followed successively by sore throat, chest pain, gastrointestinal symptoms, rash, minor hemorrhage, subconjunctival injection, and neck and facial swelling over the first week of illness. No major hemorrhage was noted. Neurological signs were sometimes seen in the late stages. Shock and multi-organ system failure, often with evidence of disseminated intravascular coagulopathy, ensued in the second week, with death in four of the five cases. Distinctive treatment components of the one surviving patient included rapid commencement of the antiviral drug ribavirin and administration of HMG-CoA reductase inhibitors (statins), N-acetylcysteine, and recombinant factor VIIa.Lujo virus causes a clinical syndrome remarkably similar to Lassa fever. Considering the high case-fatality and significant logistical impediments to controlled treatment efficacy trials for viral hemorrhagic fever, it is both logical and ethical to explore the use of the various compounds used in the treatment of the surviving case reported here in future outbreaks. Clinical observations should be systematically recorded to facilitate objective evaluation of treatment efficacy. Due to the risk of secondary transmission, viral hemorrhagic fever precautions should be implemented for all cases of Lujo virus infection, with specialized precautions to protect against aerosols when performing enhanced-risk procedures such as endotracheal intubation.
Project description:Lujo virus (LUJV), a new member of the family Arenaviridae and the first hemorrhagic fever-associated arenavirus from the Old World discovered in three decades, was isolated in South Africa during an outbreak of human disease characterized by nosocomial transmission and an unprecedented high case fatality rate of 80% (4/5 cases). Unbiased pyrosequencing of RNA extracts from serum and tissues of outbreak victims enabled identification and detailed phylogenetic characterization within 72 hours of sample receipt. Full genome analyses of LUJV showed it to be unique and branching off the ancestral node of the Old World arenaviruses. The virus G1 glycoprotein sequence was highly diverse and almost equidistant from that of other Old World and New World arenaviruses, consistent with a potential distinctive receptor tropism. LUJV is a novel, genetically distinct, highly pathogenic arenavirus.
Project description:Lujo virus (LUJV) is a novel member of the Arenaviridae family that was first identified in 2008 after an outbreak of severe hemorrhagic fever (HF). In what was a small but rapidly progressing outbreak, this previously unknown virus was transmitted from the critically ill index patient to 4 attending healthcare workers. Four persons died during this outbreak, for a total case fatality of 80% (4/5). The suspected rodent source of the initial exposure to LUJV remains a mystery. Because of the ease of transmission, high case fatality, and novel nature of LUJV, we sought to establish an animal model of LUJV HF. Initial attempts in mice failed, but infection of inbred strain 13/N guinea pigs resulted in lethal disease. A total of 41 adult strain 13/N guinea pigs were infected with either wild-type LUJV or a full-length recombinant LUJV. Results demonstrated that strain 13/N guinea pigs provide an excellent model of severe and lethal LUJV HF that closely resembles what is known of the human disease. All infected animals experienced consistent weight loss (3-5% per day) and clinical illness characterized by ocular discharge, ruffled fur, hunched posture, and lethargy. Uniform lethality occurred by 11-16 days post-infection. All animals developed disseminated LUJV infection in various organs (liver, spleen, lung, and kidney), and leukopenia, lymphopenia, thrombocytopenia, coagulopathy, and elevated transaminase levels. Serial euthanasia studies revealed a temporal pattern of virus dissemination and increasing severity of disease, primarily targeting the liver, spleen, lungs, and lower gastrointestinal tract. Establishing an animal LUJV model is an important first step towards understanding the high pathogenicity of LUJV and developing vaccines and antiviral therapeutic drugs for this highly transmissible and lethal emerging pathogen.
Project description:INTRODUCTION:Seven arenaviruses cause viral hemorrhagic fever in humans: the Old World arenaviruses Lassa and Lujo, and the New World Clade B arenaviruses Machupo (MACV), Junín (JUNV), Guanarito (GTOV), Sabiá (SABV), and Chapare (CHPV). All of these viruses are Risk Group 4 biosafety pathogens. MACV causes human disease outbreak with high case-fatality rates. To date, at least 1,200 cases with ?200 fatalities have been recorded. AREAS COVERED:This review summarizes available systems and technologies for the identification of antivirals against MACV. Furthermore, the article summarizes animal models that have been used for the in vivo evaluation of novel inhibitors. The article highlights present treatments for arenaviral diseases and provides an overview of efficacious small molecules and other therapeutics reported to date. Finally, the article summarizes strategies to identify novel inhibitors for anti-arenaviral therapy. EXPERT OPINION:New high-throughput approaches to quantitate infection rates of arenaviruses, as well as viruses modified to carry reporter genes, will accelerate compound screens and drug discovery efforts. RNAi, gene expression profiling and proteomics studies will identify host targets for therapeutic intervention. New discoveries in the cell entry mechanism of MACV and other arenaviruses as well as extensive structural studies of arenaviral L and NP could facilitate the rational design of antivirals effective against all pathogenic New World arenaviruses.
Project description:Several arenaviruses are known to cause viral hemorrhagic fever (VHF) in sub-Saharan Africa and South America, where VHF is a major public health and medical concern. The biosafety level 4 categorization of these arenaviruses restricts their use and has impeded biological studies, including therapeutic drug and/or vaccine development. Due to difficulties associated with handling live viruses, pseudotype viruses, which transiently bear arenavirus envelope proteins based on vesicular stomatitis virus (VSV) or retrovirus, have been developed as surrogate virus systems. Here, we report the development of a pseudotype VSV bearing each envelope protein of various species of arenaviruses (AREpv), including the newly identified Lujo virus (LUJV) and Chapare virus. Pseudotype arenaviruses generated in 293T cells exhibited high infectivity in various mammalian cell lines. The infections by New World and Old World AREpv were dependent on their receptors (human transferrin receptor 1 [hTfR1] and ?-dystroglycan [?DG], respectively). However, infection by pseudotype VSV bearing the LUJV envelope protein (LUJpv) occurred independently of hTfR1 and ?DG, indicating that LUJpv utilizes an unidentified receptor. The pH-dependent endocytosis of AREpv was confirmed by the use of lysosomotropic agents. The fusion of cells expressing these envelope proteins, except for those expressing the LUJV envelope protein, was induced by transient treatment at low pH values. LUJpv infectivity was inhibited by U18666A, a cholesterol transport inhibitor. Furthermore, the infectivity of LUJpv was significantly decreased in the Niemann-Pick C1 (NPC1)-deficient cell line, suggesting the necessity for NPC1 activity for efficient LUJpv infection.LUJV is a newly identified arenavirus associated with a VHF outbreak in southern Africa. Although cell entry for many arenaviruses has been studied, cell entry for LUJV has not been characterized. In this study, we found that LUJpv utilizes neither ?DG nor hTfR1 as a receptor and found unique characteristics of LUJV glycoprotein in membrane fusion and cell entry. Proper exclusion of cholesterol or some kinds of lipids may play important roles in LUJpv cell entry.
Project description:Arenaviruses cause fatal hemorrhagic disease in humans. Old World arenavirus glycoproteins (GPs) mainly engage ?-dystroglycan as a cell-surface receptor, while New World arenaviruses hijack transferrin receptor. However, the Lujo virus (LUJV) GP does not cluster with New or Old World arenaviruses. Using a recombinant vesicular stomatitis virus containing LUJV GP as its sole attachment and fusion protein (VSV-LUJV), we demonstrate that infection is independent of known arenavirus receptor genes. A genome-wide haploid genetic screen identified the transmembrane protein neuropilin 2 (NRP2) and tetraspanin CD63 as factors for LUJV GP-mediated infection. LUJV GP binds the N-terminal domain of NRP2, while CD63 stimulates pH-activated LUJV GP-mediated membrane fusion. Overexpression of NRP2 or its N-terminal domain enhances VSV-LUJV infection, and cells lacking NRP2 are deficient in wild-type LUJV infection. These findings uncover this distinct set of host cell entry factors in LUJV infection and are attractive focus points for therapeutic intervention.
Project description:Lassa virus (LASV) poses a significant public health problem within the regions of Lassa fever endemicity in Western Africa. LASV infects several hundred thousand individuals yearly, and a considerable number of Lassa fever cases are associated with high morbidity and lethality. No approved LASV vaccine is available, and current therapy is limited to an off-label usage of ribavirin that is only partially effective and associated with significant side effects. The impact of Lassa fever on human health, together with the limited existing countermeasures, highlights the importance of developing effective vaccines against LASV. Here, we present the development and characterization of a recombinant LASV (rLASV) vaccine candidate [rLASV(IGR/S-S)], which is based on the presence of the noncoding intergenic region (IGR) of the small (S) genome segment (S-IGR) in both large (L) and S LASV segments. In cultured cells, rLASV(IGR/S-S) was modestly less fit than wild-type rLASV (rLASV-WT). rLASV(IGR/S-S) was highly attenuated in guinea pigs, and a single subcutaneous low dose of the virus completely protected against otherwise lethal infection with LASV-WT. Moreover, rLASV(IGR/S-S) was genetically stable during serial passages in cultured cells. These findings indicate that rLASV(IGR/S-S) can be developed into a LASV live-attenuated vaccine (LAV) that has the same antigenic composition as LASV-WT and a well-defined mechanism of attenuation that overcomes concerns about increased virulence that could be caused by genetic changes in the LAV during multiple rounds of multiplication.IMPORTANCE Lassa virus (LASV), the causative agent of Lassa fever, infects several hundred thousand people in Western Africa, resulting in many lethal Lassa fever cases. No U.S. Food and Drug Administration-licensed countermeasures are available to prevent or treat LASV infection. We describe the generation of a novel LASV live-attenuated vaccine candidate rLASV(IGR/S-S), which is based on the replacement of the large genomic segment noncoding intergenic region (IGR) with that of the small genome segment. rLASV(IGR/S-S) is less fit in cell culture than wild-type virus and does not cause clinical signs in inoculated guinea pigs. Importantly, rLASV(IGR/S-S) protects immunized guinea pigs against an otherwise lethal exposure to LASV.
Project description:Machupo virus (MACV) is a New World (NW) arenavirus and causative agent of Bolivian hemorrhagic fever (HF). Here, we identified a variant of MACV strain Carvallo termed Car91 that was attenuated in guinea pigs. Infection of guinea pigs with an earlier passage of Carvallo, termed Car68, resulted in a lethal disease with a 63% mortality rate. Sequencing analysis revealed that compared to Car68, Car91 had a 35 nucleotide (nt) deletion and a point mutation within the L-segment intergenic region (IGR), and three silent changes in the polymerase gene that did not impact amino acid coding. No changes were found on the S-segment. Because it was apathogenic, we determined if Car91 could protect guinea pigs against Guanarito virus (GTOV), a distantly related NW arenavirus. While naïve animals succumbed to GTOV infection, 88% of the Car91-exposed guinea pigs were protected. These findings indicate that attenuated MACV vaccines can provide heterologous protection against NW arenaviruses. The disruption in the L-segment IGR, including a single point mutant and 35 nt partial deletion, were the only major variance detected between virulent and avirulent isolates, implicating its role in attenuation. Overall, our data support the development of live-attenuated arenaviruses as broadly protective pan-arenavirus vaccines.
Project description:Rift Valley fever virus (RVFV) is a mosquito-transmitted virus from the <i>Bunyaviridae</i> family that causes high rates of mortality and morbidity in humans and ruminant animals. Previous studies indicated that DEAD-box helicase 17 (DDX17) restricts RVFV replication by recognizing two primary non-coding RNAs in the S-segment of the genome: the intergenic region (IGR) and 5' non-coding region (NCR). However, we lack molecular insights into the direct binding of DDX17 with RVFV non-coding RNAs and information on the unwinding of both non-coding RNAs by DDX17. Therefore, we performed an extensive biophysical analysis of the DDX17 helicase domain (DDX17<sub>135-555</sub>) and RVFV non-coding RNAs, IGR and 5' NCR. The homogeneity studies using analytical ultracentrifugation indicated that DDX17<sub>135-555</sub>, IGR, and 5' NCR are pure. Next, we performed small-angle X-ray scattering (SAXS) experiments, which suggested that DDX17 and both RNAs are homogenous as well. SAXS analysis also demonstrated that DDX17 is globular to an extent, whereas the RNAs adopt an extended conformation in solution. Subsequently, microscale thermophoresis (MST) experiments were performed to investigate the direct binding of DDX17 to the non-coding RNAs. The MST experiments demonstrated that DDX17 binds with the IGR and 5' NCR with a dissociation constant of 5.77 ± 0.15 µM and 9.85 ± 0.11 µM, respectively. As DDX17<sub>135-555</sub> is an RNA helicase, we next determined if it could unwind IGR and NCR. We developed a helicase assay using MST and fluorescently-labeled oligos, which suggested DDX17<sub>135-555</sub> can unwind both RNAs. Overall, our study provides direct evidence of DDX17<sub>135-555</sub> interacting with and unwinding RVFV non-coding regions.