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. 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.

Single dose rVSVΔG-JUNVGP vaccine protects guinea pigs against lethal Junin virus challenge

Junin virus (JUNV) is a pathogen of biodefense importance due to its potential for aerosol transmission and mortality rates reaching 30%. Currently, there are no JUNV vaccines licensed by the United States Food and Drug Administration (FDA) for at-risk individuals. A vaccine based on recombinant vesicular stomatitis virus (rVSV) has been effectively used to prevent Ebola virus disease in humans. Here, we evaluated the protective efficacy of a rVSV expressing the JUNV glycoprotein (rVSVΔG-JUNVGP) in a guinea pig model of lethal JUNV disease. Two groups of guinea pigs, one prime and one prime-boost, were vaccinated with rVSVΔG-JUNVGP; six control animals remained unvaccinated. Survival for prime and prime-boost vaccinated animals was 100% while the challenge virus was uniformly lethal in all control animals. Animals in both vaccine groups developed robust, high avidity IgG antibody titers post-vaccination as well as detectable neutralizing antibodies while control animals failed to develop detectable antibody responses. This study demonstrates for the first time that rVSV expressing the JUNV GP fully protects guinea pigs from lethal JUNV challenge with a single injection vaccine.

Development of a Reverse Genetic System to Generate Recombinant Chimeric Tacaribe Virus that Expresses Junín Virus Glycoproteins

Mammarenaviruses are enveloped and segmented negative-stranded RNA viruses that comprise several pathogenic members associated with severe human hemorrhagic fevers. Tacaribe virus (TCRV) is the prototype for the New World group of mammarenaviruses and is not only naturally attenuated but also phylogenetically and antigenically related to all South American pathogenic mammarenaviruses, particularly the Junín virus (JUNV), which is the etiological agent of Argentinian hemorrhagic fever (AHF). Moreover, since TCRV protects guinea pigs and non-human primates from lethal challenges with pathogenic strains of JUNV, it has already been considered as a potential live-attenuated virus vaccine candidate against AHF. Here, we report the development of a reverse genetic system that relies on T7 polymerase-driven intracellular expression of the complementary copy (antigenome) of both viral S and L RNA segments. Using this approach, we successfully recovered recombinant TCRV (rTCRV) that displayed growth properties resembling those of authentic TCRV. We also generated a chimeric recombinant TCRV expressing the JUNV glycoproteins, which propagated similarly to wild-type rTCRV. Moreover, a controlled modification within the S RNA 5′ non-coding terminal sequence diminished rTCRV propagation in a cell-type dependent manner, giving rise to new perspectives where the incorporation of additional attenuation markers could contribute to develop safe rTCRV-based vaccines against pathogenic mammarenaviruses.

Human BST-2/tetherin inhibits Junin virus release from host cells and its inhibition is partially counteracted by viral nucleoprotein

Bone marrow stromal cell antigen-2 (BST-2), also known as tetherin, is an interferon-inducible membrane-associated protein. It effectively targets enveloped viruses at the release step of progeny viruses from host cells, thereby restricting the further spread of viral infection. Junin virus (JUNV) is a member of Arenaviridae, which causes Argentine haemorrhagic fever that is associated with a high rate of mortality. In this study, we examined the effect of human BST-2 on the replication and propagation of JUNV. The production of JUNV Z-mediated virus-like particles (VLPs) was significantly inhibited by over-expression of BST-2. Electron microscopy analysis revealed that BST-2 functions by forming a physical link that directly retains VLPs on the cell surface. Infection using JUNV showed that infectious JUNV production was moderately inhibited by endogenous or exogenous BST-2. We also observed that JUNV infection triggers an intense interferon response, causing an upregulation of BST-2, in infected cells. However, the expression of cell surface BST-2 was reduced upon infection. Furthermore, the expression of JUNV nucleoprotein (NP) partially recovered VLP production from BST-2 restriction, suggesting that the NP functions as an antagonist against antiviral effect of BST-2. We further showed that JUNV NP also rescued the production of Ebola virus VP40-mediated VLP from BST-2 restriction as a broad spectrum BST-2 antagonist. To our knowledge, this is the first report showing that an arenavirus protein counteracts the antiviral function of BST-2.

Type I interferon underlies severe disease associated with Junín virus infection in mice

Junín virus (JUNV) is one of five New World mammarenaviruses (NWMs) that causes fatal hemorrhagic disease in humans and is the etiological agent of Argentine hemorrhagic fever (AHF). The pathogenesis underlying AHF is poorly understood; however, a prolonged, elevated interferon-α (IFN-α) response is associated with a negative disease outcome. A feature of all NWMs that cause viral hemorrhagic fever is the use of human transferrin receptor 1 (hTfR1) for cellular entry. Here, we show that mice expressing hTfR1 develop a lethal disease course marked by an increase in serum IFN-α concentration when challenged with JUNV. Further, we provide evidence that the type I IFN response is central to the development of severe JUNV disease in hTfR1 mice. Our findings identify hTfR1-mediated entry and the type I IFN response as key factors in the pathogenesis of JUNV infection in mice.

The Glycoprotein of the Live-Attenuated Junin Virus Vaccine Strain Induces Endoplasmic Reticulum Stress and Forms Aggregates prior to Degradation in the Lysosome

ABSTRACT Argentine hemorrhagic fever is a potentially lethal disease that is caused by Junin virus (JUNV). There are currently around 5 million individuals at risk of infection within regions of endemicity in Argentina. The live attenuated vaccine strain Candid #1 (Can) is approved for use in regions of endemicity and has substantially decreased the number of annual Argentine hemorrhagic fever (AHF) cases. The glycoprotein (GPC) gene is primarily responsible for attenuation of the Can strain, and we have shown that the absence of an N-linked glycosylation motif in the subunit G1 of the glycoprotein complex of Can, which is otherwise present in the wild-type pathogenic JUNV, causes GPC retention in the endoplasmic reticulum (ER). Here, we show that Can GPC aggregates in the ER of infected cells, forming incorrect cross-chain disulfide bonds, which results in impaired GPC processing into G1 and G2. The GPC fails to cleave into its G1 and G2 subunits and is targeted for degradation within lysosomes. Cells infected with the wild-type Romero (Rom) strain do not produce aggregates that are observed in Can infection, and the stress on the ER remains minimal. While the mutation of the N-linked glycosylation motif (T168A) is primarily responsible for the formation of aggregates, other mutations within G1 that occurred earlier in the passage history of the Can strain also contribute to aggregation of the GPC within the ER. IMPORTANCE The development of vaccines and therapeutics to combat viral hemorrhagic fevers remains a top priority within the Implementation Plan of the U.S. Department of Health and Human Services Public Health Emergency Medical Countermeasures Enterprise. The Can strain, derived from the pathogenic XJ strain of JUNV, has been demonstrated to be both safe and protective against AHF. While the vaccine strain is approved for use in regions of endemicity within Argentina, the mechanisms of Can attenuation have not been elucidated. A better understanding of the viral genetic determinants of attenuation will improve our understanding of the mechanisms contributing to disease pathogenesis and provide critical information for the rational design of live attenuated vaccine candidates for other viral hemorrhagic fevers.