Treatment of highly virulent mammarenavirus infections-status quo and future directions.

INTRODUCTION: Mammarenaviruses are negative-sense bisegmented enveloped RNA viruses that are endemic in Africa, the Americas, and Europe. Several are highly virulent, causing acute human diseases associated with high case fatality rates, and are considered to be significant with respect to public health impact or bioterrorism threat. AREAS COVERED: This review summarizes the status quo of treatment development, starting with drugs that are in advanced stages of evaluation in early clinical trials, followed by promising candidate medical countermeasures emerging from bench analyses and investigational animal research. EXPERT OPINION: Specific therapeutic treatments for diseases caused by mammarenaviruses remain limited to the off-label use of ribavirin and transfusion of convalescent sera. Progress in identifying novel candidate medical countermeasures against mammarenavirus infection has been slow in part because of the biosafety and biosecurity requirements. However, novel methodologies and tools have enabled increasingly efficient high-throughput molecular screens of regulatory-agency-approved small-molecule drugs and led to the identification of several compounds that could be repurposed for the treatment of infection with several mammarenaviruses. Unfortunately, most of them have not yet been evaluated in vivo. The most promising treatment under development is a monoclonal antibody cocktail that is protective against multiple lineages of the Lassa virus in nonhuman primate disease models.

Molecular Epidemiology of Mayaro Virus among Febrile Patients, Roraima State, Brazil, 2018-2021.

We detected Mayaro virus (MAYV) in 3.4% (28/822) of febrile patients tested during 2018-2021 from Roraima State, Brazil. We also isolated MAYV strains and confirmed that these cases were caused by genotype D. Improved surveillance is needed to better determine the burden of MAYV in the Amazon Region.

Development of reverse genetics system for Guanarito virus: substitution of E1497K in the L protein of Guanarito virus S-26764 strain changes plaque phenotype and growth kinetics.

Guanarito virus (GTOV) is the causative agent of Venezuelan hemorrhagic fever. GTOV belongs to the genus Mammarenavirus, family Arenaviridae and has been classified as a Category A bioterrorism agent by the United States Centers for Disease Control and Prevention. Despite being a high-priority agent, vaccines and drugs against Venezuelan hemorrhagic fever are not available. GTOV S-26764, isolated from a non-fatal human case, produces an unclear cytopathic effect (CPE) in Vero cells, posing a significant obstacle to research and countermeasure development efforts. Vero cell-adapted GTOV S-26764 generated in this study produced clear CPE and demonstrated rapid growth and high yield in Vero cells compared to the original GTOV S-26764. We developed a reverse genetics system for GTOV to study amino acid changes acquired through Vero cell adaptation and leading to virus phenotype changes. The results demonstrated that E1497K in the L protein was responsible for the production of clear plaques as well as enhanced viral RNA replication and transcription efficiency. Vero cell-adapted GTOV S-26764, capable of generating CPE, will allow researchers to easily perform neutralization assays and anti-drug screening against GTOV. Moreover, the developed reverse genetics system will accelerate vaccine and antiviral drug development.IMPORTANCEGuanarito virus (GTOV) is a rodent-borne virus. GTOV causes fever, prostration, headache, arthralgia, cough, sore throat, nausea, vomiting, diarrhea, epistaxis, bleeding gums, menorrhagia, and melena in humans. The lethality rate is 23.1% or higher. Vero cell-adapted GTOV S-26764 shows a clear cytopathic effect (CPE), whereas the parental virus shows unclear CPE in Vero cells. We generated a reverse genetics system to rescue recombinant GTOVs and found that E1497K in the L protein was responsible for the formation of clear plaques as well as enhanced viral RNA replication and transcription efficiency. This reverse genetic system will accelerate vaccine and antiviral drug developments, and the findings of this study contribute to the understanding of the function of GTOV L as an RNA polymerase.

Clonally Expanded Virus-Specific CD8 T Cells Acquire Diverse Transcriptional Phenotypes During Acute, Chronic, and Latent Infections.

CD8+ T cells play a crucial role in the control and resolution of viral infections and can adopt a wide range of phenotypes and effector functions depending on the inflammatory context and the duration and extent of antigen exposure. Similarly, viral infections can exert diverse selective pressures on populations of clonally related T cells. Technical limitations have nevertheless made it challenging to investigate the relationship between clonal selection and transcriptional phenotypes of virus-specific T cells. We therefore performed single-cell T cell receptor (TCR) repertoire and transcriptome sequencing of virus-specific CD8 T cells in murine models of acute, chronic and latent infection. We observed clear infection-specific populations corresponding to memory, effector, exhausted, and inflationary phenotypes. We further uncovered a mouse-specific and polyclonal T cell response, despite all T cells sharing specificity to a single viral epitope, which was accompanied by stereotypic TCR germline gene usage in all three infection types. Persistent antigen exposure during chronic and latent viral infections resulted in a higher proportion of clonally expanded T cells relative to acute infection. We furthermore observed a relationship between transcriptional heterogeneity and clonal expansion for all three infections, with highly expanded clones having distinct transcriptional phenotypes relative to less expanded clones. Together our work relates clonal selection to gene expression in the context of viral infection and further provides a dataset and accompanying software for the immunological community.

Different but Not Unique: Deciphering the Immunity of the Jamaican Fruit Bat by Studying Its Viriome.

A specialized and fine-tuned immune response of bats upon infection with viruses is believed to provide the basis for a "friendly" coexistence with these pathogens, which are often lethal for humans and other mammals. First insights into the immunity of bats suggest that bats have evolved to possess their own strategies to cope with viral infections. Yet, the molecular details for this innocuous coexistence remain poorly described and bat infection models are the key to unveiling these secrets. In Jamaican fruit bats (Artibeus jamaicensis), a New World bat species, infection experiments with its (putative) natural viral pathogens Tacaribe virus (TCRV), rabies virus (RABV), and the bat influenza A virus (IAV) H18N11, have contributed to an accurate, though still incomplete, representation of the bat-imposed immunity. Surprisingly, though many aspects of their innate and adaptive immune responses differ from that of the human immune response, such as a contraction of the IFN locus and reduction in the number of immunoglobulin subclasses, variations could also be observed between Jamaican fruit bats and other bat species.

Persistent RNA virus infection is short-lived at the single-cell level but leaves transcriptomic footprints.

Several RNA viruses can establish life-long persistent infection in mammalian hosts, but the fate of individual virus-infected cells remains undefined. Here we used Cre recombinase-encoding lymphocytic choriomeningitis virus to establish persistent infection in fluorescent cell fate reporter mice. Virus-infected hepatocytes underwent spontaneous noncytolytic viral clearance independently of type I or type II interferon signaling or adaptive immunity. Viral clearance was accompanied by persistent transcriptomic footprints related to proliferation and extracellular matrix remodeling, immune responses, and metabolism. Substantial overlap with persistent epigenetic alterations in HCV-cured patients suggested a universal RNA virus-induced transcriptomic footprint. Cell-intrinsic clearance occurred in cell culture, too, with sequential infection, reinfection cycles separated by a period of relative refractoriness to infection. Our study reveals that systemic persistence of a prototypic noncytolytic RNA virus depends on continuous spread and reinfection. Yet undefined cell-intrinsic mechanisms prevent viral persistence at the single-cell level but give way to profound transcriptomic alterations in virus-cleared cells.

Isolation of Reconstructed Functional Ribonucleoprotein Complexes of Machupo Virus.

Arenaviruses initiate infection by delivering a transcriptionally competent ribonucleoprotein (RNP) complex into the cytosol of host cells. The arenavirus RNP consists of the large (L) RNA-dependent RNA polymerase (RdRP) bound to a nucleoprotein (NP)-encapsidated genomic RNA (viral RNA [vRNA]) template. During transcription and replication, L must transiently displace RNA-bound NP to allow for template access into the RdRP active site. Concomitant with RNA replication, new subunits of NP must be added to the nascent complementary RNAs (cRNA) as they emerge from the product exit channel of L. Interactions between L and NP thus play a central role in arenavirus gene expression. We developed an approach to purify recombinant functional RNPs from mammalian cells in culture using a synthetic vRNA and affinity-tagged L and NP. Negative-stain electron microscopy of purified RNPs revealed they adopt diverse and flexible structures, like RNPs of other Bunyavirales members. Monodispersed L-NP and trimeric ring-like NP complexes were also obtained in excess of flexible RNPs, suggesting that these heterodimeric structures self-assemble in the absence of suitable RNA templates. This work allows for further biochemical analysis of the interaction between arenavirus L and NP proteins and provides a framework for future high-resolution structural analyses of this replication-associated complex. IMPORTANCE Arenaviruses are rodent-borne pathogens that can cause severe disease in humans. All arenaviruses begin the infection cycle with delivery of the virus replication machinery into the cytoplasm of the host cell. This machinery consists of an RNA-dependent RNA polymerase-which copies the viral genome segments and synthesizes all four viral mRNAs-bound to the two nucleoprotein-encapsidated genomic RNAs. How this complex assembles remains a mystery. Our findings provide direct evidence for the formation of diverse intracellular arenavirus replication complexes using purification strategies for the polymerase, nucleoprotein, and genomic RNA of Machupo virus, which causes Bolivian hemorrhagic fever in humans. We demonstrate that the polymerase and nucleoprotein assemble into higher-order structures within cells, providing a model for the molecular events of arenavirus RNA synthesis. These findings provide a framework for probing the architectures and functions of the arenavirus replication machinery and thus advancing antiviral strategies targeting this essential complex.

Screening and Identification of Lujo Virus Inhibitors Using a Recombinant Reporter Virus Platform.

Lujo virus (LUJV), a highly pathogenic arenavirus, was first identified in 2008 in Zambia. To aid the identification of effective therapeutics for LUJV, we developed a recombinant reporter virus system, confirming reporter LUJV comparability with wild-type virus and its utility in high-throughput antiviral screening assays. Using this system, we evaluated compounds with known and unknown efficacy against related arenaviruses, with the aim of identifying LUJV-specific and potential new pan-arenavirus antivirals. We identified six compounds demonstrating robust anti-LUJV activity, including several compounds with previously reported activity against other arenaviruses. These data provide critical evidence for developing broad-spectrum antivirals against high-consequence arenaviruses.

Prevention of CD8 T Cell Deletion during Chronic Viral Infection.

During chronic viral infections, CD8 T cells rapidly lose antiviral and immune-stimulatory functions in a sustained program termed exhaustion. In addition to this loss of function, CD8 T cells with the highest affinity for viral antigen can be physically deleted. Consequently, treatments designed to restore function to exhausted cells and control chronic viral replication are limited from the onset by the decreased breadth of the antiviral T cell response. Yet, it remains unclear why certain populations of CD8 T cells are deleted while others are preserved in an exhausted state. We report that CD8 T cell deletion during chronic viral infection can be prevented by therapeutically lowering viral replication early after infection. The initial resistance to deletion enabled long-term maintenance of antiviral cytolytic activity of the otherwise deleted high-affinity CD8 T cells. In combination with decreased virus titers, CD4 T cell help and prolonged interactions with costimulatory molecules B7-1/B7-2 were required to prevent CD8 T cell deletion. Thus, therapeutic strategies to decrease early virus replication could enhance virus-specific CD8 T cell diversity and function during chronic infection.

Slow viral propagation during initial phase of infection leads to viral persistence in mice.

Immune evasion of pathogens can modify the course of infection and impact viral persistence and pathology. Here, using different strains of the lymphocytic choriomeningitis virus (LCMV) model system, we show that slower propagation results in limited type I interferon (IFN-I) production and viral persistence. Specifically, cells infected with LCMV-Docile exhibited reduced viral replication when compared to LCMV-WE and as a consequence, infection with LCMV-Docile resulted in reduced activation of bone marrow derived dendritic cells (BMDCs) and IFN-I production in vitro in comparison with LCMV-WE. In vivo, we observed a reduction of IFN-I, T cell exhaustion and viral persistence following infection of LCMV-Docile but not LCMV-WE. Mechanistically, block of intracellular protein transport uncovered reduced propagation of LCMV-Docile when compared to LCMV-WE. This reduced propagation was critical in blunting the activation of the innate and adaptive immune system. When mice were simultaneously infected with LCMV-Docile and LCMV-WE, immune function was restored and IFN-I production, T cell effector functions as well as viral loads were similar to that of mice infected with LCMV-WE alone. Taken together, this study suggests that reduced viral propagation can result in immune evasion and viral persistence.

Effector and stem-like memory cell fates are imprinted in distinct lymph node niches directed by CXCR3 ligands.

T cells dynamically interact with multiple, distinct cellular subsets to determine effector and memory differentiation. Here, we developed a platform to quantify cell location in three dimensions to determine the spatial requirements that direct T cell fate. After viral infection, we demonstrated that CD8+ effector T cell differentiation is associated with positioning at the lymph node periphery. This was instructed by CXCR3 signaling since, in its absence, T cells are confined to the lymph node center and alternatively differentiate into stem-like memory cell precursors. By mapping the cellular sources of CXCR3 ligands, we demonstrated that CXCL9 and CXCL10 are expressed by spatially distinct dendritic and stromal cell subsets. Unlike effector cells, retention of stem-like memory precursors in the paracortex is associated with CCR7 expression. Finally, we demonstrated that T cell location can be tuned, through deficiency in CXCL10 or type I interferon signaling, to promote effector or stem-like memory fates.

BACH2 enforces the transcriptional and epigenetic programs of stem-like CD8+ T cells.

During chronic infection and cancer, a self-renewing CD8+ T cell subset maintains long-term immunity and is critical to the effectiveness of immunotherapy. These stem-like CD8+ T cells diverge from other CD8+ subsets early after chronic viral infection. However, pathways guarding stem-like CD8+ T cells against terminal exhaustion remain unclear. Here, we show that the gene encoding transcriptional repressor BACH2 is transcriptionally and epigenetically active in stem-like CD8+ T cells but not terminally exhausted cells early after infection. BACH2 overexpression enforced stem-like cell fate, whereas BACH2 deficiency impaired stem-like CD8+ T cell differentiation. Single-cell transcriptomic and epigenomic approaches revealed that BACH2 established the transcriptional and epigenetic programs of stem-like CD8+ T cells. In addition, BACH2 suppressed the molecular program driving terminal exhaustion through transcriptional repression and epigenetic silencing. Thus, our study reveals a new pathway that enforces commitment to stem-like CD8+ lineage and prevents an alternative terminally exhausted cell fate.

Inhibition of Arenaviruses by Combinations of Orally Available Approved Drugs.

Neglected diseases caused by arenaviruses such as Lassa virus (LASV) and filoviruses like Ebola virus (EBOV) primarily afflict resource-limited countries, where antiviral drug development is often minimal. Previous studies have shown that many approved drugs developed for other clinical indications inhibit EBOV and LASV and that combinations of these drugs provide synergistic suppression of EBOV, often by blocking discrete steps in virus entry. We hypothesize that repurposing of combinations of orally administered approved drugs provides effective suppression of arenaviruses. In this report, we demonstrate that arbidol, an approved influenza antiviral previously shown to inhibit EBOV, LASV, and many other viruses, inhibits murine leukemia virus (MLV) reporter viruses pseudotyped with the fusion glycoproteins (GPs) of other arenaviruses (Junin virus [JUNV], lymphocytic choriomeningitis virus [LCMV], and Pichinde virus [PICV]). Arbidol and other approved drugs, including aripiprazole, amodiaquine, sertraline, and niclosamide, also inhibit infection of cells by infectious PICV, and arbidol, sertraline, and niclosamide inhibit infectious LASV. Combining arbidol with aripiprazole or sertraline results in the synergistic suppression of LASV and JUNV GP-bearing pseudoviruses. This proof-of-concept study shows that arenavirus infection in vitro can be synergistically inhibited by combinations of approved drugs. This approach may lead to a proactive strategy with which to prepare for and control known and new arenavirus outbreaks.

DAPK1 (death associated protein kinase 1) mediates mTORC1 activation and antiviral activities in CD8+ T cells.

Mechanistic target of rapamycin complex 1 (mTORC1) regulates CD8+ T-cell differentiation and function. Despite the links between PI3K-AKT and mTORC1 activation in CD8+ T cells, the molecular mechanism underlying mTORC1 activation remains unclear. Here, we show that both the kinase activity and the death domain of DAPK1 are required for maximal mTOR activation and CD8+ T-cell function. We found that TCR-induced activation of calcineurin activates DAPK1, which subsequently interacts with TSC2 via its death domain and phosphorylates TSC2 to mediate mTORC1 activation. Furthermore, both the kinase domain and death domain of DAPK1 are required for CD8+ T-cell antiviral responses in an LCMV infection model. Together, our data reveal a novel mechanism of mTORC1 activation that mediates optimal CD8+ T-cell function and antiviral activity.

BH3-only sensors Bad, Noxa and Puma are Key Regulators of Tacaribe virus-induced Apoptosis.

Pathogenicity often differs dramatically among even closely related arenavirus species. For instance, Junín virus (JUNV), the causative agent of Argentine hemorrhagic fever (AHF), is closely related to Tacaribe virus (TCRV), which is normally avirulent in humans. While little is known about how host cell pathways are regulated in response to arenavirus infection, or how this contributes to virulence, these two viruses have been found to differ markedly in their ability to induce apoptosis. However, details of the mechanism(s) governing the apoptotic response to arenavirus infections are unknown. Here we confirm that TCRV-induced apoptosis is mitochondria-regulated, with associated canonical hallmarks of the intrinsic apoptotic pathway, and go on to identify the pro- and anti-apoptotic Bcl-2 factors responsible for regulating this process. In particular, levels of the pro-apoptotic BH3-only proteins Noxa and Puma, as well as their canonical transcription factor p53, were strongly increased. Interestingly, TCRV infection also led to the accumulation of the inactive phosphorylated form of another pro-apoptotic BH3-only protein, Bad (i.e. as phospho-Bad). Knockout of Noxa or Puma suppressed apoptosis in response to TCRV infection, whereas silencing of Bad increased apoptosis, confirming that these factors are key regulators of apoptosis induction in response to TCRV infection. Further, we found that while the highly pathogenic JUNV does not induce caspase activation, it still activated upstream pro-apoptotic factors, consistent with current models suggesting that JUNV evades apoptosis by interfering with caspase activation through a nucleoprotein-mediated decoy function. This new mechanistic insight into the role that individual BH3-only proteins and their regulation play in controlling apoptotic fate in arenavirus-infected cells provides an important experimental framework for future studies aimed at dissecting differences in the apoptotic responses between arenaviruses, their connection to other cell signaling events and ultimately the relationship of these processes to pathogenesis.

Virulent infection of outbred Hartley guinea pigs with recombinant Pichinde virus as a surrogate small animal model for human Lassa fever.

Arenaviruses, such as Lassa virus (LASV), can cause severe and fatal hemorrhagic fevers (e.g., Lassa fever, LF) in humans with no vaccines or therapeutics. Research on arenavirus-induced hemorrhagic fevers (AHFs) has been hampered by the highly virulent nature of these viral pathogens, which require high biocontainment laboratory, and the lack of an immune-competent small animal model that can recapitulate AHF disease and pathological features. Guinea pig infected with Pichinde virus (PICV), an arenavirus that does not cause disease in humans, has been established as a convenient surrogate animal model for AHFs as it can be handled in a conventional laboratory. The PICV strain P18, derived from sequential passaging of the virus 18 times in strain 13 inbred guinea pigs, causes severe febrile illness in guinea pigs that is reminiscent of lethal LF in humans. As inbred guinea pigs are not readily available and are difficult to maintain, outbred Hartley guinea pigs have been used but they show a high degree of disease heterogeneity upon virulent P18 PICV infection. Here, we describe an improved outbred guinea-pig infection model using recombinant rP18 PICV generated by reverse genetics technique followed by plaque purification, which consistently shows >90% mortality and virulent infection. Comprehensive virological, histopathological, and immunohistochemical analyses of the rP18-virus infected animals show similar features of human LASV infection. Our data demonstrate that this improved animal model can serve as a safe, affordable, and convenient surrogate small animal model for studying human LF pathogenesis and for evaluating efficacy of preventative or therapeutic approaches.

Distinct Molecular Mechanisms of Host Immune Response Modulation by Arenavirus NP and Z Proteins.

Endemic to West Africa and South America, mammalian arenaviruses can cross the species barrier from their natural rodent hosts to humans, resulting in illnesses ranging from mild flu-like syndromes to severe and fatal haemorrhagic zoonoses. The increased frequency of outbreaks and associated high fatality rates of the most prevalent arenavirus, Lassa, in West African countries, highlights the significant risk to public health and to the socio-economic development of affected countries. The devastating impact of these viruses is further exacerbated by the lack of approved vaccines and effective treatments. Differential immune responses to arenavirus infections that can lead to either clearance or rapid, widespread and uncontrolled viral dissemination are modulated by the arenavirus multifunctional proteins, NP and Z. These two proteins control the antiviral response to infection by targeting multiple cellular pathways; and thus, represent attractive targets for antiviral development to counteract infection. The interplay between the host immune responses and viral replication is a key determinant of virus pathogenicity and disease outcome. In this review, we examine the current understanding of host immune defenses against arenavirus infections and summarise the host protein interactions of NP and Z and the mechanisms that govern immune evasion strategies.

Profiling Virus-Specific Tcf1+ T Cell Repertoires During Acute and Chronic Viral Infection.

CD8 T cells play a crucial role in providing protection from viral infections. It has recently been established that a subset of CD8 T cells expressing Tcf1 are responsible for sustaining exhausted T cells during chronic lymphocytic choriomeningitis virus (LCMV) infection. Many of these studies, however, have been performed using T cell receptor (TCR) transgenic mice, in which CD8 T cells express a monoclonal TCR specific for the LCMV glycoprotein. To investigate whether the Tcf1+ and Tcf1- repertoires are naturally composed of similar or different clones in wild-type mice exposed to acute or chronic LCMV infection, we performed TCR repertoire sequencing of virus-specific CD8 T cells, including Tcf1+ and Tcf1- populations. Our analysis revealed that the Tcf1+ TCR repertoire is maintained at an equal or higher degree of clonal diversity despite harboring fewer cells. Additionally, within the same animal, there was extensive clonal overlap between the Tcf1+ and Tcf1- repertoires in both chronic and acute LCMV infection. We could further detect these virus-specific clones in longitudinal blood samples earlier in the infection. With respect to common repertoire parameters (clonal overlap, germline gene usage, and clonal expansion), we found minor differences between the virus-specific TCR repertoire of acute and chronic LCMV infection 40 days post infection. Overall, our results indicate that the Tcf1+ population emerging during chronic LCMV infection is not clonally distinct from the Tcf1- population, supporting the notion that the Tcf1+ pool is indeed a fuel for the more exhausted Tcf1- population within the heterogenous repertoire of LCMV-specific CD8 T cells.