Preclinical evaluation of two phylogenetically distant arenavirus vectors for the development of novel immunotherapeutic combination strategies for cancer treatment.

BACKGROUND: Engineered arenavirus vectors have recently been developed to leverage the body's immune system in the fight against chronic viral infections and cancer. Vectors based on Pichinde virus (artPICV) and lymphocytic choriomeningitis virus (artLCMV) encoding a non-oncogenic fusion protein of human papillomavirus (HPV)16 E6 and E7 are currently being tested in patients with HPV16+ cancer, showing a favorable safety and tolerability profile and unprecedented expansion of tumor-specific CD8+ T cells. Although the strong antigen-specific immune response elicited by artLCMV vectors has been demonstrated in several preclinical models, PICV-based vectors are much less characterized. METHODS: To advance our understanding of the immunobiology of these two vectors, we analyzed and compared their individual properties in preclinical in vivo and in vitro systems. Immunogenicity and antitumor effect of intratumoral or intravenous administration of both vectors, as well as combination with NKG2A blockade, were evaluated in naïve or TC-1 mouse tumor models. Flow cytometry, Nanostring, and histology analysis were performed to characterize the tumor microenvironment (TME) and T-cell infiltrate following treatment. RESULTS: Despite being phylogenetically distant, both vectors shared many properties, including preferential infection and activation of professional antigen-presenting cells, and induction of potent tumor-specific CD8+ T-cell responses. Systemic as well as localized treatment induced a proinflammatory shift in the TME, promoting the infiltration of inducible T cell costimulator (ICOS)+CD8+ T cells capable of mediating tumor regression and prolonging survival in a TC-1 mouse tumor model. Still, there was evidence of immunosuppression built-up over time, and increased expression of H2-T23 (ligand for NKG2A T cell inhibitory receptor) following treatment was identified as a potential contributing factor. NKG2A blockade improved the antitumor efficacy of artARENA vectors, suggesting a promising new combination approach. This demonstrates how detailed characterization of arenavirus vector-induced immune responses and TME modulation can inform novel combination therapies. CONCLUSIONS: The artARENA platform represents a strong therapeutic vaccine approach for the treatment of cancer. The induced antitumor immune response builds the backbone for novel combination therapies, which warrant further investigation.

Novel Oliveros-like Clade C Mammarenaviruses from Rodents in Argentina, 1990-2020.

Following an Argentine Hemorrhagic Fever (AHF) outbreak in the early 1990s, a rodent survey for Junín virus, a New World Clade B arenavirus, in endemic areas of Argentina was conducted. Since 1990, INEVH has been developing eco-epidemiological surveillance of rodents, inside and outside the Argentine Hemorrhagic Fever endemic area. Samples from rodents captured between 1993 and 2019 that were positive for Arenavirus infection underwent Sanger and unbiased, Illumina-based high-throughput sequencing, which yielded 5 complete and 88 partial Mammarenaviruses genomes. Previously, 11 genomes representing four species of New World arenavirus Clade C existed in public records. This work has generated 13 novel genomes, expanding the New World arenavirus Clade C to 24 total genomes. Additionally, two genomes exhibit sufficient genetic diversity to be considered a new species, as per ICTV guidelines (proposed name Mammarenavirus vellosense). The 13 novel genomes exhibited reassortment between the small and large segments in New World Mammarenaviruses. This work demonstrates that Clade C Mammarenavirus infections circulate broadly among Necromys species in the Argentine Hemorrhagic Fever endemic area; however, the risk for Clade C Mammarenavirus human infection is currently unknown.

Luminescent reporter cells enable the identification of broad-spectrum antivirals against emerging viruses.

The emerging viruses SARS-CoV-2 and arenaviruses cause severe respiratory and hemorrhagic diseases, respectively. The production of infectious particles of both viruses and virus spread in tissues requires cleavage of surface glycoproteins (GPs) by host proprotein convertases (PCs). SARS-CoV-2 and arenaviruses rely on GP cleavage by PCs furin and subtilisin kexin isozyme-1/site-1 protease (SKI-1/S1P), respectively. We report improved luciferase-based reporter cell lines, named luminescent inducible proprotein convertase reporter cells that we employ to monitor PC activity in its authentic subcellular compartment. Using these sensor lines we screened a small compound library in high-throughput manner. We identified 23 FDA-approved small molecules, among them monensin which displayed broad activity against furin and SKI-1/S1P. Monensin inhibited arenaviruses and SARS-CoV-2 in a dose-dependent manner. We observed a strong reduction in infectious particle release upon monensin treatment with little effect on released genome copies. This was reflected by inhibition of SARS-CoV-2 spike processing suggesting the release of immature particles. In a proof of concept experiment using human precision cut lung slices, monensin potently inhibited SARS-CoV-2 infection, evidenced by reduced infectious particle release. We propose that our PC sensor pipeline is a suitable tool to identify broad-spectrum antivirals with therapeutic potential to combat current and future emerging viruses.

Epidemiology and Genomic characteristics of arenavirus in rodents from the southeast coast of P.R. China.

BACKGROUND: Wenzhou virus (WENV), a member of the Mammarenavirus genus in the Arenaviridae family, has been detected in wild rodents from eight provinces in China, including Zhejiang, Shandong, Hainan, Xinjiang, Hunan, Guangdong, Yunnan, and Jiangxi provinces, and some countries from Southeast Asia. The IgG-antibodies of WENV have been detected in both healthy populations and patients with unknown fever and respiratory symptoms. However, the potential harmfulness of WENV to humans has been underestimated due to mild symptoms after infection, similar to respiratory diseases. Thus, it is imperative to enhance the surveillance of WENV in wild rodents, particularly Rattus norvegicus, and continuously monitor its prevalence. RESULTS: From 2017 to 2021, a total of 390 wild rodents were collected from six provinces in the eastern and southern coastal areas, containing nine species of rats. Samples of each tissue were collected, and PCR amplified for identification. Four R. norvegicus samples were detected to be WENV-positive. No genomic sequence of WENV was detected in Rattus flavipectus, Rattus losea, Suncus murinus, Apodemus agrarius, Mus musculus, Microtus fortis, Micromys minutus, and Niviventer niviventer from Jiangsu, Zhejiang, Fujian, Hainan, Guangdong and Guangxi provinces. Three genomic sequences were identified to be WENV by phylogenetic analysis. The full-length sequences of HAIKOU-40 were amplified in R. norvegicus from Hainan, which showed a close relationship to Wufeng/ WFS, sharing 84.5-89.4% homology at the nucleotide level and 91.6-98.9% homology at the amino acid level. Phylogenetic analysis revealed that HAIKOU-40 formed an Asia-specific cluster with all WENVs and Loie River mammarenavirus (LORV), provisionally named Asian ancestry. This cluster has diverged earlier from the remaining mammarenavirus. The sequences obtained in Xiamen, Fujian province showed more than 90% nucleotide identities with WENV, which may be a strain of WENV. Additionally, the sequence of Wuxi-87 which was a positive sequence detected in Wuxi, Jiangsu province exhibited 83% nucleotide identity with Lassa virus (LASV). Further efforts will be made to isolate and identify this virus strain, verify the relationship between Wuxi-87 and LASV, and confirm whether R. norvegicus is a new host of LASV. CONCLUSIONS: In this study, we conducted a systematic examination of the prevalence of WENV among rodents on the southeast coast of China. Additionally, we characterized the genome of a newly discovered WENV strain, that confirmed the role of R. norvegicus in the transmission of WENV. This highlights the importance of investigating the prevalence of WENV in both wild rodents and humans.

[Recent advances in the development of vaccines against hemorrhagic fevers caused by arenaviruses]. / Les fièvres hémorragiques causées par les arénavirus : de récentes avancées vaccinales.

Arenaviruses are a global threat, causing thousands of deaths each year in several countries around the world. Despite strong efforts in the development of vaccine candidates, vaccines against Lassa fever or Bolivian and Venezuelan hemorrhagic fevers are yet to be licensed for a use in humans. In this synthesis, we present the arenaviruses causing fatal diseases in humans and the main vaccine candidates that have been developed over the past decades with an emphasis on the measles-Lassa vaccine, the first Lassa vaccine ever tested in humans, and on the MOPEVAC platform that can potentially be used as a pan-arenavirus vaccine platform.

Title: Les fièvres hémorragiques causées par les arénavirus : de récentes avancées vaccinales. Abstract: Le développement de vaccins contre les arénavirus est un enjeu global. En effet, plusieurs milliers de personnes meurent chaque année de la fièvre de Lassa en Afrique occidentale et les virus Machupo, Guanarito ou Chapare continuent de ré-émerger en Amérique du Sud. Pourtant, il n'existe à ce jour aucun vaccin validé pour une utilisation dans l'espèce humaine pour lutter contre ces arénavirus. Dans cette synthèse, nous présentons les différents arénavirus causant des maladies mortelles chez l'espèce humaine et les principaux candidats vaccins développés au cours des dernières décennies contre ces virus. Nous décrivons plus particulièrement le vaccin rougeole-Lassa, premier vaccin contre la fièvre de Lassa à avoir été testé dans l'espèce humaine, et la plateforme MOPEVAC qui permet de générer avec succès des vaccins mono- ou multivalents contre potentiellement tous les arénavirus pathogènes connus.

Hemostasis defects underlying the hemorrhagic syndrome caused by mammarenaviruses in a cynomolgus macaque model.

Viral hemorrhagic fevers (HF) are a group of acute febrile diseases with high mortality rates. Although hemostatic dysfunction appears to be a major determinant of the severity of the disease, it is still unclear what pathogenic mechanisms lead to it. In clinical studies it is found that arenaviruses, such as Lassa, Machupo, and Guanarito viruses cause HF that vary in symptoms and biological alterations. In this study we aimed to characterize the hemostatic dysfunction induced by arenaviral HF to determine its implication in the severity of the disease and to elucidate the origin of this syndrome. We found that lethal infection with Machupo, Guanarito, and Lassa viruses is associated with cutaneomucosal, cerebral, digestive, and pulmonary hemorrhages. The affected animals developed a severe alteration of the coagulation system, which was concomitant with acute hepatitis, minor deficit of hepatic factor synthesis, presence of a plasmatic inhibitor of coagulation, and dysfunction of the fibrinolytic system. Despite signs of increased vascular permeability, endothelial cell infection was not a determinant factor of the hemorrhagic syndrome. There were also alterations of the primary hemostasis during lethal infection, with moderate to severe thrombocytopenia and platelet dysfunction. Finally, we show that lethal infection is accompanied by a reduced hematopoietic potential of the bone marrow. This study provides an unprecedented characterization of the hemostasis defects induced by several highly pathogenic arenaviruses.

Effects of the Natural Flavonoid Quercetin on Arenavirus Junín Infection.

There is no specific chemotherapy approved for the treatment of pathogenic arenaviruses that cause severe hemorrhagic fever (HF) in the population of endemic regions in America and Africa. The present study reports the effects of the natural flavonoid quercetin (QUER) on the infection of A549 and Vero cells with Junín virus (JUNV), agent of the Argentine HF. By infectivity assays, a very effective dose-dependent reduction of JUNV multiplication was shown by cell pretreatment at 2-6 h prior to the infection at non-cytotoxic concentrations, with 50% effective concentration values in the range of 6.1-7.5 µg/mL. QUER was also active by post-infection treatment but with minor efficacy. Mechanistic studies indicated that QUER mainly affected the early steps of virus adsorption and internalization in the multiplication cycle of JUNV. Treatment with QUER blocked the phosphorylation of Akt without changes in the total protein expression, detected by Western blot, and the consequent perturbation of the PI3K/Akt pathway was also associated with the fluorescence redistribution from membrane to cytoplasm of TfR1, the cell receptor recognized by JUNV. Then, it appears that the cellular antiviral state, induced by QUER treatment, leads to the prevention of JUNV entry into the cell.

Fusogenic structural changes in arenavirus glycoproteins are associated with viroporin activity.

Many enveloped viruses enter host cells by fusing with acidic endosomes. The fusion activity of multiple viral envelope glycoproteins does not generally affect viral membrane permeability. However, fusion induced by the Lassa virus (LASV) glycoprotein complex (GPc) is always preceded by an increase in viral membrane permeability and the ensuing acidification of the virion interior. Here, systematic investigation of this LASV fusion phenotype using single pseudovirus tracking in live cells reveals that the change in membrane barrier function is associated with the fusogenic conformational reorganization of GPc. We show that a small-molecule fusion inhibitor or mutations that impair viral fusion by interfering with GPc refolding into the post-fusion structure prevent the increase in membrane permeability. We find that the increase in virion membrane permeability occurs early during endosomal maturation and is facilitated by virus-cell contact. This increase is observed using diverse arenavirus glycoproteins, whether presented on lentivirus-based pseudoviruses or arenavirus-like particles, and in multiple different cell types. Collectively, these results suggest that conformational changes in GPc triggered by low pH and cell factor binding are responsible for virion membrane permeabilization and acidification of the virion core prior to fusion. We propose that this viroporin-like activity may augment viral fusion and/or post-fusion steps of infection, including ribonucleoprotein release into the cytoplasm.

Sphingosine Kinases Promote Ebola Virus Infection and Can Be Targeted to Inhibit Filoviruses, Coronaviruses, and Arenaviruses Using Late Endocytic Trafficking to Enter Cells.

Entry of enveloped viruses in host cells requires the fusion of viral and host cell membranes, a process that is facilitated by viral fusion proteins protruding from the viral envelope. These viral fusion proteins need to be triggered by host factors, and for some viruses, this event occurs inside endosomes and/or lysosomes. Consequently, these 'late-penetrating viruses' must be internalized and delivered to entry-conducive intracellular vesicles. Because endocytosis and vesicular trafficking are tightly regulated cellular processes, late-penetrating viruses also depend on specific host proteins for efficient delivery to the site of fusion, suggesting that these could be targeted for antiviral therapy. In this study, we investigated a role for sphingosine kinases (SKs) in viral entry and found that chemical inhibition of sphingosine kinase 1 (SK1) and/or SK2 and knockdown of SK1/2 inhibited entry of Ebola virus (EBOV) into host cells. Mechanistically, inhibition of SK1/2 prevented EBOV from reaching late-endosomes and lysosomes that contain the EBOV receptor, Niemann Pick C1 (NPC1). Furthermore, we present evidence that suggests that the trafficking defect caused by SK1/2 inhibition occurs independently of sphingosine-1-phosphate (S1P) signaling through cell-surface S1P receptors. Lastly, we found that chemical inhibition of SK1/2 prevents entry of other late-penetrating viruses, including arenaviruses and coronaviruses, and inhibits infection by replication-competent EBOV and SARS-CoV-2 in Huh7.5 cells. In sum, our results highlight an important role played by SK1/2 in endocytic trafficking, which can be targeted to inhibit entry of late-penetrating viruses and could serve as a starting point for the development of broad-spectrum antiviral therapeutics.

Management of Hemorrhagic Fever Viruses: Intervention of Natural and Synthetic Products.

BACKGROUND: Viral hemorrhagic fevers (VHFs) are a group of clinical syndromes caused by several different RNA virus families, including several members of the arenavirus, bunyavirus, filovirus, and flavivirus families. VHFs have high mortality rates, and they have been associated with vascular permeability, malaise, fever, variable degrees of hemorrhage, reduced plasma volume, and coagulation abnormalities. To treat such conditions, antigen-presenting cells target dysregulated immune reactions and productive infections. Monocytes and macrophages produce inflammatory cytokines that damage adaptive immunity, while infected dendritic cells fail to mature correctly, compromising adaptive immunity. Inflammation and uncontrolled virus replication are associated with vascular leakage and coagulopathy. OBJECTIVE: VHF infects both humans and animals and if not treated, causes hemorrhagic manifestations and lethal platelet dysfunction. Besides pharmacological and immunological solutions, the intervention of natural products for VHF management is of great interest. In this review, we gathered current data about the effectiveness of natural products for VHF management. METHODS: Data were extracted from Scopus, Google Scholar, PubMed, and Cochrane library in terms of clinical and animal studies published in English between 1981 to February 2022. RESULTS: Several plants from diverse families and species were identified with antiviral activity against VHF. The combination of botanical therapeutics and multitarget synergistic therapeutic effects is now the widely accepted explanation for the treatment of VHF. Most of these herbal therapeutics have shown promising immunomodulatory effects in vivo and in vitro VHF models. They can probably modulate the immune system in VHF-infected subjects mainly by interfering with certain inflammatory mediators involved in various infectious diseases. CONCLUSION: Natural, in particular, herbal sources can be valuable for the management of various VHFs and their related complications.

Design, Synthesis, and Biological Evaluation of Benzimidazole Derivatives as Potential Lassa Virus Inhibitors.

The Lassa virus (LASV) causes Lassa fever, a highly infectious and lethal agent of acute viral hemorrhagic fever. At present, there are still no effective treatments available, creating an urgent need to develop novel therapeutics. Some benzimidazole compounds targeting the arenavirus envelope glycoprotein complex (GPC) are promising inhibitors of LASV. In this study, we synthesized two series of LASV inhibitors based on the benzimidazole structure. Lentiviral pseudotypes bearing the LASV GPC were established to identify virus entry inhibitors. Surface plasmon resonance (SPR) was further used to verify the binding activities of the potential compounds. Compounds 7d-Z, 7h-Z, 13c, 13d, and 13f showed relatively excellent antiviral activities with IC50 values ranging from 7.58 to 15.46 nM and their SI values above 1251. These five representative compounds exhibited stronger binding affinity with low equilibrium dissociation constants (KD < 8.25 × 10-7 M) in SPR study. The compound 7h-Z displayed the most potent antiviral activity (IC50 = 7.58 nM) with a relatively high SI value (2496), which could be further studied as a lead compound. The structure-activity relationship indicated that the compounds with lipophilic and spatially larger substituents might possess higher antiviral activity and a much larger safety margin. This study will provide some good guidance for the development of highly active compounds with a novel skeleton against LASV.

A dsRNA-binding mutant reveals only a minor role of exonuclease activity in interferon antagonism by the arenavirus nucleoprotein.

The arenavirus nucleoprotein (NP) plays an important role in the virus' ability to block interferon (IFN) production, and its exonuclease function appears to contribute to this activity. However, efforts to analyze this contribution are complicated by the functional overlap between the exonuclease active site and a neighboring region involved in IKKε-binding and subsequent inhibition of IRF3 activation, which also plays an important role in IFN production. To circumvent this issue, we mutated a residue located away from the active site that is involved in binding of the dsRNA substrate being targeted for exonuclease digestion, i.e. H426A. We found that expression of Tacaribe virus (TCRV) NP containing this RNA-binding H426A mutation was still able to efficiently block IFN-ß promoter activity in response to Sendai virus infection, despite being strongly impaired in its exonuclease activity. This was in contrast to a conventional exonuclease active site mutant (E388A), which was impaired with respect to both exonuclease activity and IFN antagonism. Importantly, growth of a recombinant virus encoding the RNA-binding mutation (rTCRV-H426A) was similar to wild-type in IFN-deficient cells, unlike the active site mutant (rTCRV-E388A), which was already markedly impaired in these cells. Further, in IFN-competent cells, the TCRV-H426A RNA-binding mutant showed more robust growth and delayed IFN-ß mRNA upregulation compared to the TCRV-E388A active site mutant. Taken together, this novel mutational approach, which allows us to now dissect the different contributions of the NP exonuclease activity and IKKε-binding/IRF3 inhibition to IFN antagonism, clearly suggests that conventional exonuclease mutants targeting the active site overestimate the contribution of the exonuclease function, and that rather other IFN antagonistic functions of NP play the dominant role in IFN-antagonism.

Serological evidence of arenavirus circulation in wild rodents from central-west, southeast, and south regions of Brazil, 2002-2006.

Viral hemorrhagic fevers caused by arenaviruses are severe zoonotic diseases. In reservoirs, the presence of antibodies may indicate viral circulation in a population of a specific region, and these data can be used as an indicator for further investigations by molecular techniques. The present study aimed to detect the presence of arenavirus antibodies in wild rodents captured from 1998 to 2008 during epidemiological surveillance activities. A retrospective analysis of 2243 wild rodent blood samples using a broad cross-reactive in-house developed enzyme-linked immunosorbent assay (ELISA) revealed a 0.44% (10/2243) positive rate in wild rodents, which included Necromys lasiurus (6/1012), Calomys callosus (2/94), and Akodon sp. (2/273) species. These rodents were captured between 2002 to 2006 in Campo Alegre de Goiás/GO, Bodoquena/MS, Nuporanga/SP, and Mogi das Cruzes/SP. Our findings suggest the sylvatic circulation of arenavirus among wild rodents in the southeast region of Brazil. However, future virological and molecular studies are necessary to confirm the viral presence in these regions.

Highly Diverse Arenaviruses in Neotropical Bats, Brazil.

We detected arenavirus RNA in 1.6% of 1,047 bats in Brazil that were sampled during 2007-2011. We identified Tacaribe virus in 2 Artibeus sp. bats and a new arenavirus species in Carollia perspicillata bats that we named Tietê mammarenavirus. Our results suggest that bats are an underrecognized arenavirus reservoir.

Understanding Host-Virus Interactions: Assessment of Innate Immune Responses in Mastomys natalensis Cells after Arenavirus Infection.

Mastomys natalensis is the natural host of various arenaviruses, including the human-pathogenic Lassa virus. Homologous arenaviruses, defined here as those having M. natalensis as a natural host, can establish long-lasting infection in M. natalensis, while these animals rapidly clear arenaviruses having another rodent species as a natural host (heterologous viruses). Little is known about the mechanisms behind the underlying arenavirus-host barriers. The innate immune system, particularly the type I interferon (IFN) response, might play a role. In this study, we developed and validated RT-PCR assays to analyse the expression of M. natalensis interferon-stimulated genes (ISGs). We then used these assays to study if homologous and heterologous viruses induce different IFN responses in M. natalensis cells. Infection experiments were performed with the homologous Lassa and Morogoro viruses and the related but heterologous Mobala virus. Compared to the direct induction with IFN or Poly(I:C), arenaviruses generally induced a weak IFN response. However, the ISG-expression profiles of homologous and heterologous viruses were similar. Our data indicate that, at least in M. natalensis cells, the IFN system is not a major factor in the virus-host barrier for arenaviruses. Our system provides a valuable tool for future in vivo investigation of arenavirus host restrictions at the level of the innate immune response.

Potential and action mechanism of favipiravir as an antiviral against Junin virus.

Favipiravir is a nucleoside analogue that inhibits the replication and transcription of a broad spectrum of RNA viruses, including pathogenic arenaviruses. In this study, we isolated a favipiravir-resistant mutant of Junin virus (JUNV), which is the causative agent of Argentine hemorrhagic fever, and analyzed the antiviral mechanism of favipiravir against JUNV. Two amino acid substitutions, N462D in the RNA-dependent RNA polymerase (RdRp) and A168T in the glycoprotein precursor GPC, were identified in the mutant. GPC-A168T substitution enhanced the efficiency of JUNV internalization, which explains the robust replication kinetics of the mutant in the virus growth analysis. Although RdRp-N462D substitution did not affect polymerase activity levels in a minigenome system, comparisons of RdRp error frequencies showed that the virus with RdRp-D462 possessed a significantly higher fidelity. Our next generation sequence (NGS) analysis showed a gradual accumulation of both mutations as we passaged the virus in presence of favipiravir. We also provided experimental evidence for the first time that favipiravir inhibited JUNV through the accumulation of transition mutations, confirming its role as a purine analogue against arenaviruses. Moreover, we showed that treatment with a combination of favipiravir and either ribavirin or remdesivir inhibited JUNV replication in a synergistic manner, blocking the generation of the drug-resistant mutant. Our findings provide new insights for the clinical management and treatment of Argentine hemorrhagic fever.

Generation of Reporter-Expressing New World Arenaviruses: A Systematic Comparison.

Replication-competent reporter-expressing viruses are crucial tools in molecular virology with applications that range from antiviral screening to live-cell imaging of protein spatiotemporal dynamics. However, there is currently little information available regarding viable strategies to develop reporter-expressing arenaviruses. To address this, we used Tacaribe virus (TCRV), an apathogenic BSL2 arenavirus, to assess the feasibility of different reporter expression approaches. We first generated trisegmented TCRV viruses with either the glycoprotein (GP) or nucleoprotein (NP) replaced by a reporter (GFP, mCherry, or nanoluciferase). These viruses were all viable, but showed marked differences in brightness and attenuation. Next, we generated terminal fusions with each of the TCRV proteins (i.e., NP, GP, polymerase (L), matrix protein (Z)) either with or without a T2A self-cleavage site. We tested both the function of the reporter-fused proteins alone, and the viability of corresponding recombinant TCRVs. We successfully rescued viruses with both direct and cleavable reporter fusions at the C-terminus of Z, as well as cleavable N-terminal fusions with NP. These viruses all displayed detectable reporter activity, but were also moderately attenuated. Finally, reporter proteins were inserted into a flexible hinge region within L. These viruses were also viable and showed moderate attenuation; however, reporter expression was only detectable for the luminescent virus. These strategies provide an exciting range of new tools for research into the molecular biology of TCRV that can likely also be adapted to other arenaviruses.

The Virus-Host Interplay in Junín Mammarenavirus Infection.

Junín virus (JUNV) belongs to the Arenaviridae family and is the causative agent of Argentine hemorrhagic fever (AHF), a severe human disease endemic to agricultural areas in Argentina. At this moment, there are no effective antiviral therapeutics to battle pathogenic arenaviruses. Cumulative reports from recent years have widely provided information on cellular factors playing key roles during JUNV infection. In this review, we summarize research on host molecular determinants that intervene in the different stages of the viral life cycle: viral entry, replication, assembly and budding. Alongside, we describe JUNV tight interplay with the innate immune system. We also review the development of different reverse genetics systems and their use as tools to study JUNV biology and its close teamwork with the host. Elucidating relevant interactions of the virus with the host cell machinery is highly necessary to better understand the mechanistic basis beyond virus multiplication, disease pathogenesis and viral subversion of the immune response. Altogether, this knowledge becomes essential for identifying potential targets for the rational design of novel antiviral treatments to combat JUNV as well as other pathogenic arenaviruses.

Identification of potent inhibitors of arenavirus and SARS-CoV-2 exoribonucleases by fluorescence polarization assay.

Viral exoribonucleases are uncommon in the world of RNA viruses. To date, they have only been identified in the Arenaviridae and the Coronaviridae families. The exoribonucleases of these viruses play a crucial role in the pathogenicity and interplay with host innate immune response. Moreover, coronaviruses exoribonuclease is also involved in a proofreading mechanism ensuring the genetic stability of the viral genome. Because of their key roles in virus life cycle, they constitute attractive target for drug design. Here we developed a sensitive, robust and reliable fluorescence polarization assay to measure the exoribonuclease activity and its inhibition in vitro. The effectiveness of the method was validated on three different viral exoribonucleases, including SARS-CoV-2, Lymphocytic Choriomeningitis and Machupo viruses. We performed a screening of a focused library consisting of 113 metal chelators. Hit compounds were recovered with an IC50 at micromolar level. We confirmed 3 hits in SARS-CoV-2 infected Vero-E6 cells.

Infection of Human Endothelial Cells with Lassa Virus Induces Early but Transient Activation and Low Type I IFN Response Compared to the Closely-Related Nonpathogenic Mopeia Virus.

Lassa virus (LASV), an Old World arenavirus, is responsible for hemorrhagic fevers in western Africa. The privileged tropism of LASV for endothelial cells combined with a dysregulated inflammatory response are the main cause of the increase in vascular permeability observed during the disease. Mopeia virus (MOPV) is another arenavirus closely related to LASV but nonpathogenic for non-human primates (NHPs) and has never been described in humans. MOPV is more immunogenic than LASV in NHPs and in vitro in human immune cell models, with more intense type I IFN and adaptive cellular responses. Here, we compared the transcriptomic and proteomic responses of human umbilical vein endothelial cells (HUVECs) to infection with the two viruses to further decipher the mechanisms involved in their differences in immunogenicity and pathogenicity. Both viruses replicated durably and efficiently in HUVECs, but the responses they induced were strikingly different. Modest activation was observed at an early stage of LASV infection and then rapidly shut down. By contrast, MOPV induced a late but more intense response, characterized by the expression of genes and proteins mainly associated with the type I IFN response and antigen processing/presentation. Such a response is consistent with the higher immunogenicity of MOPV relative to LASV, whereas the lack of an innate response induced in HUVECs by LASV is consistent with its uncontrolled systemic dissemination through the vascular endothelium.