Advancement of vaccine candidates that demonstrate protective efficacy in screening studies necessitates detailed safety and immunogenicity investigations in pre-clinical models. A non-spreading Crimean-Congo hemorrhagic fever virus (CCHFV) viral replicon particle (VRP) vaccine was developed for single-dose administration to protect against disease. To date, several studies have supported safety, immunogenicity, and efficacy of the CCHF VRP in multiple highly sensitive murine models of lethal disease, but the VRP had yet to be evaluated in large animals. Here, we performed studies in non-human primates to further evaluate clinical utility of the VRP vaccine. Twelve adult male and female rhesus macaques were vaccinated intramuscularly and followed daily for clinical monitoring. At 3, 7, 14, 21, and 28 days post vaccination, animals were sedated for more detailed clinical assessment; for quantification of vaccine presence in blood and mucosal samples; and for evaluation of hematology, plasma inflammatory markers, and immunity. Consistent with findings in mice, vaccination was well tolerated, with no clinical alterations nor indication of vaccine spread or shedding. In addition, vaccination induced both humoral and cell-mediated responses, with immune profile and kinetics also corroborating data from small animal models. These studies provide key data in non-human primates further supporting development of the VRP for human clinical use.

Development of broad-spectrum antiviral therapies is critical for outbreak and pandemic preparedness against emerging and reemerging viruses. Viruses inducing hemorrhagic fevers cause high morbidity and mortality in humans and are associated with several recent international outbreaks, but approved therapies for treating most of these pathogens are lacking. Here, we show that 4'-fluorouridine (4'-FlU; EIDD-2749), an orally available ribonucleoside analog, has antiviral activity against multiple hemorrhagic fever viruses in cell culture, including Nipah virus, Crimean-Congo hemorrhagic fever virus, orthohantaviruses, and arenaviruses. We performed preclinical in vivo evaluation of oral 4'-FlU against two arenaviruses, Old World Lassa virus (LASV) and New World Junín virus (JUNV), in guinea pig models of lethal disease. 4'-FlU demonstrated both advantageous pharmacokinetic characteristics and high efficacy in both of these lethal disease guinea pig models. Additional experiments supported protection of the infected animals even when 4'-FlU delivery was reduced to a low dose of 0.5 milligram per kilogram. To demonstrate clinical utility, 4'-FlU treatment was evaluated when initiated late in the course of infection (12 or 9 days after infection for LASV and JUNV, respectively). Delayed treatment resulted in rapid resolution of clinical signs, demonstrating an extended window for therapeutic intervention. These data support the use of 4'-FlU as a potent and efficacious treatment against highly pathogenic arenaviruses of public health concern with a virus inhibition profile suggesting broad-spectrum utility as an orally available antiviral drug against a wide variety of viral pathogens.

Immunizing mice with Crimean-Congo hemorrhagic fever virus (CCHFV) nucleoprotein (NP), glycoprotein precursor (GPC), or with the GP38 domain of GPC, can be protective when the proteins are delivered with viral vectors or as a DNA or RNA vaccine. Subunit vaccines are a safe and cost-effective alternative to some vaccine platforms, but Gc and Gn glycoprotein subunit vaccines for CCHFV fail to protect despite eliciting high levels of neutralizing antibodies. Here, we investigated humoral and cellular immune responses and the protective efficacy of recombinant NP, GP38, and GP38 forms (GP85 and GP160) associated with the highly glycosylated mucin-like (MLD) domain, as well as the NP + GP38 combination. Vaccination with GP160, GP85, or GP38 did not confer protection, and vaccination with the MLD-associated GP38 forms blunted the humoral immune responses to GP38, worsened clinical chemistry, and increased viral RNA in the blood compared to the GP38 vaccination. In contrast, NP vaccination conferred 100% protection from lethal outcome and was associated with mild clinical disease, while the NP + GP38 combination conferred even more robust protection by reducing morbidity compared to mice receiving NP alone. Thus, recombinant CCHFV NP alone is a promising vaccine candidate conferring 100% survival against heterologous challenge. Moreover, incorporation of GP38 should be considered as it further enhances subunit vaccine efficacy by reducing morbidity in surviving animals.

Crimean-Congo hemorrhagic fever virus (CCHFV) can cause severe human disease and is considered a WHO priority pathogen due to the lack of efficacious vaccines and antivirals. A CCHF virus replicon particle (VRP) has previously shown protective efficacy in a lethal Ifnar(-/-) mouse model when administered as a single dose at least 3 days prior to challenge. Here, we determine that non-specific immune responses are not sufficient to confer short-term protection, since Lassa virus VRP vaccination 3 days prior to CCHFV challenge was not protective. We also investigate how CCHF VRP vaccination confers protective efficacy by examining viral kinetics, histopathology, clinical analytes and immunity early after challenge (3 and 6 days post infection) and compare to unvaccinated controls. We characterize how these effects differ based on vaccination period and correspond to previously reported CCHF VRP-mediated protection. Vaccinating Ifnar(-/-) mice with CCHF VRP 28, 14, 7, or 3 days prior to challenge, all known to confer complete protection, significantly reduced CCHFV viral load, mucosal shedding, and markers of clinical disease, with greater reductions associated with longer vaccination periods. Interestingly, there were no significant differences in innate immune responses, T cell activation, or antibody titers after challenge between groups of mice vaccinated a week or more before challenge, but higher anti-NP antibody avidity and effector function (ADCD) were positively associated with longer vaccination periods. These findings support the importance of antibody-mediated responses in VRP vaccine-mediated protection against CCHFV infection.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne nairovirus with a wide geographic spread that can cause severe and lethal disease. No specific medical countermeasures are approved to combat this illness. The CCHFV L protein contains an ovarian tumor (OTU) domain with a cysteine protease thought to modulate cellular immune responses by removing ubiquitin and ISG15 post-translational modifications from host and viral proteins. Viral deubiquitinases like CCHFV OTU are attractive drug targets, as blocking their activity may enhance cellular immune responses to infection, and potentially inhibit viral replication itself. We previously demonstrated that the engineered ubiquitin variant CC4 is a potent inhibitor of CCHFV replication in vitro. A major challenge of the therapeutic use of small protein inhibitors such as CC4 is their requirement for intracellular delivery, e.g., by viral vectors. In this study, we examined the feasibility of in vivo CC4 delivery by a replication-deficient recombinant adenovirus (Ad-CC4) in a lethal CCHFV mouse model. Since the liver is a primary target of CCHFV infection, we aimed to optimize delivery to this organ by comparing intravenous (tail vein) and intraperitoneal injection of Ad-CC4. While tail vein injection is a traditional route for adenovirus delivery, in our hands intraperitoneal injection resulted in higher and more widespread levels of adenovirus genome in tissues, including, as intended, the liver. However, despite promising in vitro results, neither route of in vivo CC4 treatment resulted in protection from a lethal CCHFV infection.

Nipah virus (NiV) is a highly pathogenic paramyxovirus. The Syrian hamster model recapitulates key features of human NiV disease and is a critical tool for evaluating antivirals and vaccines. Here we describe longitudinal humoral immune responses in NiV-infected Syrian hamsters. Samples were obtained 1-28 days after infection and analyzed by ELISA, neutralization, and Fc-mediated effector function assays. NiV infection elicited robust antibody responses against the nucleoprotein and attachment glycoprotein. Levels of neutralizing antibodies were modest and only detectable in surviving animals. Fc-mediated effector functions were mostly observed in nucleoprotein-targeting antibodies. Antibody levels and activities positively correlated with challenge dose.

Crimean-Congo hemorrhagic fever virus (CCHFV; family Nairoviridae) is a tick-borne pathogen that frequently causes lethal disease in humans. CCHFV has a wide geographic distribution, and cases have been reported in Africa, Asia, the Middle East, and Europe. Availability of a safe and efficacious vaccine is critical for restricting outbreaks and preventing disease in endemic countries. We previously developed a virus-like replicon particle (VRP) vaccine that provides complete protection against homologous and heterologous lethal CCHFV challenge in mice after a single dose. However, the immune responses induced by this vaccine are not well characterized, and correlates of protection remain unknown. Here we comprehensively characterized the kinetics of cell-mediated and humoral immune responses in VRP-vaccinated mice, and demonstrate that they predominantly target the nucleoprotein (NP). NP antibodies are not associated with protection through neutralizing activity, but VRP vaccination results in NP antibodies possessing Fc-mediated antibody effector functions, such as complement activation (ADCD) and antibody-mediated cellular phagocytosis (ADCP). This suggests that Fc-mediated effector functions may contribute to this vaccine's efficacy.

Nipah virus (NiV) causes a highly lethal disease in humans who present with acute respiratory or neurological signs. No vaccines against NiV have been approved to date. Here, we report on the clinical impact of a novel NiV-derived nonspreading replicon particle lacking the fusion (F) protein gene (NiVΔF) as a vaccine in three small animal models of disease. A broad antibody response was detected that included immunoglobulin G (IgG) and IgA subtypes with demonstrable Fc-mediated effector function targeting multiple viral antigens. Single-dose intranasal vaccination up to 3 days before challenge prevented clinical signs and reduced virus levels in hamsters and immunocompromised mice; decreases were seen in tissues and mucosal secretions, critically decreasing potential for virus transmission. This virus replicon particle system provides a vital tool to the field and demonstrates utility as a highly efficacious and safe vaccine candidate that can be administered parenterally or mucosally to protect against lethal Nipah disease.

Development of lethal models of Ebola virus disease has been achieved by the serial passage of virus isolates from human cases in mice and guinea pigs. Use of mice infected with non-adapted virus has been limited due to the absence of overt clinical disease. In recent years, newly recognized sequelae identified in human cases has highlighted the importance of continued investigations of non-lethal infection both in humans and animal models. Here, we revisit the use of rodent-adapted and non-adapted Ebola virus (EBOV) variants in mice to investigate infection tolerance and future utility of these models in pathogenesis and therapeutic intervention studies. We found that like non-adapted wild-type EBOV, guinea pig-adapted EBOV results in widespread tissue infection, variably associated with tissue pathology, and alterations in clinical and immunological analytes in the absence of overt disease. Notably, infection with either non-lethal variant does not greatly differ from lethal mouse-adapted EBOV until near the time end-point criteria are reached in these mice, supporting use of these models of virus tolerance for continued investigations of non-lethal infection and sequelae.

ABSTRACTLassa fever (LF) is endemic to broad regions of West Africa. Infection with Lassa virus (LASV), the etiologic agent of LF, results in a spectrum of clinical signs in humans, including severe and lethal hemorrhagic disease. Person-to-person transmission occurs through direct contact with body fluids or contaminated bedding and clothing. To investigate transmission risk in acute LASV infection, we evaluated viral RNA and infectious virus obtained from conjunctival, nasal, oral, genital, and rectal swab specimens from guinea pigs modeling lethal and non-lethal LF. Viral RNA and infectious virus were detected in all specimen types beginning 8 days post infection, prior to onset of fever. In the pre-clinical and clinical period, virus was isolated from a subset of nasal, oral, genital, and rectal swabs, and from all conjunctival swabs. Overall, conjunctival and nasal specimens most frequently yielded infectious virus. These findings indicate mucosal transmission risk based on virus isolation from various sites early in infection and support potential utility of minimally invasive specimen evaluation by RT-qPCR for LASV diagnostics.

Defective interfering particles (DIs) contain a considerably smaller genome than the parental virus but retain replication competency. As DIs can directly or indirectly alter propagation kinetics of the parental virus, they offer a novel approach to antiviral therapy, capitalizing on knowledge from natural infection. However, efforts to translate in vitro inhibition to in vivo screening models remain limited. We investigated the efficacy of virus-like particles containing DI genomes (therapeutic infectious particles [TIPs]) in the Syrian hamster model of lethal Nipah virus (NiV) disease. We found that coadministering a high dose of TIPs intraperitoneally with virus challenge improved clinical course and reduced lethality. To mimic natural exposure, we also evaluated lower-dose TIP delivery and virus challenge intranasally, finding equally efficacious reduction in disease severity and overall lethality. Eliminating TIP replicative capacity decreased efficacy, suggesting protection via direct inhibition. These data provide evidence that TIP-mediated treatment can confer protection against disease and lethal outcome in a robust animal NiV model, supporting further development of TIP treatment for NiV and other high-consequence pathogens. IMPORTANCE Here, we demonstrate that treatment with defective interfering particles (DIs), a natural by-product of viral infection, can significantly improve the clinical course and outcome of viral disease. When present with their parental virus, DIs can directly or indirectly alter viral propagation kinetics and exert potent inhibitory properties in cell culture. We evaluated the efficacy of a selection of virus-like particles containing DI genomes (TIPs) delivered intranasally in a lethal hamster model of Nipah virus disease. We demonstrate significantly improved clinical outcomes, including reduction in both lethality and the appearance of clinical signs. This work provides key efficacy data in a robust model of Nipah virus disease to support further development of TIP-mediated treatment against high-consequence viral pathogens.

Lassa virus (LASV) causes mild to severe hemorrhagic fever disease in humans. Strain 13/N guinea pigs are highly susceptible to infection with LASV strain Josiah (clade IV), providing a critical model system for therapeutics and vaccine development. To develop additional models of disease, we detail the clinical course in guinea pigs infected with 5 geographically and genetically diverse LASV strains. Two of the developed models (LASV clades II and III) were then used to evaluate efficacy of a virus replicon particle (VRP) vaccine against heterologous LASV challenge, demonstrating complete protection against clinical disease after a single vaccination dose.

Post-translational modification of host and viral proteins by ubiquitin and ubiquitin-like proteins plays a key role in a host's ability to mount an effective immune response. Avian species lack a ubiquitin-like protein found in mammals and other non-avian reptiles; interferon stimulated gene product 15 (ISG15). ISG15 serves as a messenger molecule and can be conjugated to both host and viral proteins leading them to be stabilized, degraded, or sequestered. Structurally, ISG15 is comprised of a tandem ubiquitin-like domain (Ubl), which serves as the motif for post-translational modification. The 2'-5' oligoadenylate synthetase-like proteins (OASL) also encode two Ubl domains in series near its C-terminus which binds OASL to retinoic acid inducible gene-I (RIG-I). This protein-protein interaction increases the sensitivity of RIG-I and results in an enhanced production of type 1 interferons and a robust immune response. Unlike human and other mammalian OASL homologues, avian OASLs terminate their tandem Ubl domains with the same LRLRGG motif found in ubiquitin and ISG15, a motif required for their conjugation to proteins. Chickens, however, lack RIG-I, raising the question of structural and functional characteristics of chicken OASL (chOASL). By investigating chOASL, the evolutionary history of viruses with deubiquitinases can be explored and drivers of species specificity for these viruses may be uncovered. Here we show that the chOASL tandem Ubl domains shares structural characteristics with mammalian ISG15, and that chOASL can oligomerize and conjugate to itself. In addition, the ISG15-like features of avian OASLs and how they impact interactions with viral deubiquitinases and deISGylases are explored.

Crimean-Congo hemorrhagic fever virus (CCHFV) causes mild to severe and fatal disease in humans. Person-to-person transmission is common, necessitating the availability of rapidly deliverable therapeutic and prophylactic interventions to mitigate CCHFV spread. Previously, we showed complete protection using one dose of a viral replicon particle (VRP) vaccine administered 28 days before CCHFV challenge. In order to determine the utility of the VRP vaccine for rapid vaccination protocols, we assessed the efficacy of such vaccination administered at various intervals relative to challenge in IFNAR(-/-) mice. Unvaccinated mice uniformly succumbed to disease by 8 days post infection (dpi). All mice vaccinated 14, 7, or 3 days prior to CCHFV challenge survived infection. Mice vaccinated -14 or -7 dpi were fully protected from clinical disease, whereas mice inoculated -3 dpi developed signs of disease prior to recovering to baseline values 5-9 dpi. These data support the utility of the VRP vaccine for modified short course vaccination protocols to protect against disease and severe outcomes.

Interferon (IFN)-stimulated gene product 15 (ISG15) is a ubiquitin-like protein critical for the control of microbial infections. ISG15 appears to serve a wide variety of functions, which regulate multiple cellular responses contributing to the development of an antiviral state. ISG15 is a versatile molecule directly modulating both host and virus protein function which regulate many signaling pathways, including its own synthesis. Here we review the various roles ISG15 plays in the antiviral immune response, and examine the mechanisms by which viruses attempt to mitigate or exploit ISG15 activity.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tri-segmented, tick-borne nairovirus that causes disease of ranging severity in humans. The CCHFV M segment encodes a complex glycoprotein precursor (GPC) that undergoes extensive endoproteolytic cleavage, giving rise to two structural proteins (Gn and Gc) required for virus attachment and entry, and to multiple non-structural proteins (NSm, GP160, GP85, and GP38). The functions of these non-structural proteins remain largely unclear. Here, we investigate the role of NSm during infection by generating a recombinant CCHFV lacking the complete NSm domain (10200NSm) and observing CCHFV NSm replication in cell lines and pathogenicity in Ifnar(-/-) mice. Our data demonstrate that the NSm domain is dispensable for viral replication in vitro, and, despite the delayed onset of clinical signs, CCHFV lacking this domain caused severe or lethal disease in infected mice.

The error-prone nature of ribonucleic acid (RNA)-dependent RNA polymerases drives the diversity of RNA virus populations. Arising within this diversity is a subset of defective viral genomes that retain replication competency, termed defective interfering (DI) genomes. These defects are caused by aberrant viral polymerase reinitiation on the same viral RNA template (deletion DI species) or the nascent RNA strand (copyback DI species). Defective interfering genomes have previously been shown to alter the dynamics of a viral population by interfering with normal virus replication and/or by stimulating the innate immune response. In this study, we investigated the ability of artificially produced DI genomes to inhibit Nipah virus (NiV), a highly pathogenic biosafety level 4 paramyxovirus. High multiplicity of infection passaging of both NiV clinical isolates and recombinant NiV in Vero cells generated an extensive DI population from which individual DIs were identified using next-generation sequencing techniques. Assays were established to generate and purify both naturally occurring and in silico-designed DIs as fully encapsidated, infectious virus-like particles termed defective interfering particles (DIPs). We demonstrate that several of these NiV DIP candidates reduced NiV titers by up to 4 logs in vitro. These data represent a proof-of-principle that a therapeutic application of DIPs to combat NiV infections may be an alternative source of antiviral control for this disease.

Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus that causes fatal encephalitis and respiratory disease in humans. There is currently no approved therapeutic for human use against NiV infection. Griffithsin (GRFT) is high-mannose oligosaccharide binding lectin that has shown in vivo broad-spectrum activity against viruses including severe acute respiratory syndrome coronavirus, human immunodeficiency virus 1, hepatitis C virus, and Japanese encephalitis virus. In this study, we evaluated the in vitro antiviral activities of GRFT and its synthetic trimeric tandemer (3mG) against NiV and other viruses from across 4 virus families. The 3mG had comparatively greater potency than GRFT against NiV due to its enhanced ability to block NiV glycoprotein-induced syncytia formation. Our initial in vivo prophylactic evaluation of an oxidation-resistant GRFT (Q-GRFT) showed significant protection against lethal NiV challenge in Syrian golden hamsters. Our results warrant further development of Q-GRFT and 3mG as potential NiV therapeutics.

Tick-borne nairoviruses (order Bunyavirales) encode an ovarian tumor domain protease (OTU) that suppresses the innate immune response by reversing the post-translational modification of proteins by ubiquitin (Ub) and interferon-stimulated gene product 15 (ISG15). Ub is highly conserved across eukaryotes, whereas ISG15 is only present in vertebrates and shows substantial sequence diversity. Prior attempts to address the effect of ISG15 diversity on viral protein-ISG15 interactions have focused on only a single species' ISG15 or a limited selection of nairovirus OTUs. To gain a more complete perspective of OTU-ISG15 interactions, we biochemically assessed the relative activities of 14 diverse nairovirus OTUs for 12 species' ISG15 and found that ISG15 activity is predominantly restricted to particular nairovirus lineages reflecting, in general, known virus-host associations. To uncover the underlying molecular factors driving OTUs affinity for ISG15, X-ray crystal structures of Kupe virus and Ganjam virus OTUs bound to sheep ISG15 were solved and compared to complexes of Crimean-Congo hemorrhagic fever virus and Erve virus OTUs bound to human and mouse ISG15, respectively. Through mutational and structural analysis seven residues in ISG15 were identified that predominantly influence ISG15 species specificity among nairovirus OTUs. Additionally, OTU residues were identified that influence ISG15 preference, suggesting the potential for viral OTUs to adapt to different host ISG15s. These findings provide a foundation to further develop research methods to trace nairovirus-host relationships and delineate the full impact of ISG15 diversity on nairovirus infection.

Crimean-Congo hemorrhagic fever virus (CCHFV, order Bunyavirales, family Nairoviridae, genus Orthonairovirus) is the tick-borne etiological agent of Crimean-Congo hemorrhagic fever (CCHF) in humans. Animals are generally susceptible to CCHFV infection but refractory to disease. Small animal models are limited to interferon-deficient mice, that develop acute fatal disease following infection. Here, using a ZsGreen1- (ZsG) expressing reporter virus (CCHFV/ZsG), we examine tissue tropism and dissemination of virus in interferon-alpha/beta receptor knock-out (Ifnar-/-) mice. We demonstrate that CCHFV/ZsG retains in vivo pathogenicity comparable to wild-type virus. Interestingly, despite high levels of viral RNA in all organs assessed, 2 distribution patterns of infection were observed by both fluorescence and immunohistochemistry (IHC), corresponding to the permissiveness of organ tissues. To further investigate viral dissemination and to temporally define cellular targets of CCHFV in vivo, mice were serially euthanized at different stages of disease. Flow cytometry was used to characterize CCHFV-associated alterations in hematopoietic cell populations and to classify infected cells in the blood, lymph node, spleen, and liver. ZsG signal indicated that mononuclear phagocytic cells in the lymphatic tissues were early targets of infection; in late-stage infection, overall, the highest levels of signal were detected in the liver, and ZsG was found in both antigen-presenting and lymphocyte cell populations.

Nipah virus ([NiV] family Paramyxoviridae, genus Henipavirus) infection can cause severe respiratory and neurological disease in humans. The pathophysiology of disease is not fully understood, and it may vary by presentation and clinical course. In this study, we investigate changes in blood chemistry in NiV-infected Syrian hamsters that survived or succumbed to disease. Increased sodium and magnesium and decreased albumin and lactate levels were detected in animals euthanized with severe clinical disease compared with mock-infected controls. When subjects were grouped by clinical syndrome, additional trends were discernable, highlighting changes associated with either respiratory or neurological disease.

In the absence of approved vaccines and therapeutics for use in humans, Nipah virus continues to cause fatal outbreaks of encephalitis and respiratory disease in Bangladesh and India on a near-annual basis. We determined that a single dose of a lipid nanoparticle nucleoside-modified messenger RNA vaccine encoding the soluble Hendra virus glycoprotein protected up to 70% of Syrian hamsters from lethal NiV challenge, despite animals having suboptimally primed immune responses before challenge. These data provide a foundation from which to optimize future messenger RNA vaccination studies against NiV and other highly pathogenic viruses.

Using a recombinant Nipah virus expressing a fluorescent protein (ZsG), we visualized virus tropism in the Syrian hamster model. We found that anatomical localization of fluorescence correlated to clinical signs; signal was primarily visualized in the respiratory tract in animals with acute-onset terminal disease, whereas central nervous system localization was seen in animals that succumbed with delayed disease onset. While polymerase chain reaction (PCR) detection corresponded well to ZsG signal, virus was only isolated from some lung, brain, liver, and kidney samples that were ZsG and/or PCR positive, and only from animals euthanized on or before 15 days post infection.

No vaccines are currently licensed to prevent Crimean-Congo hemorrhagic fever virus (CCHFV) infection, which can cause mild self-limiting clinical signs or severe, often fatal hemorrhagic fever disease. Here we continued investigations into the utility of a single-dose virus replicon particle (VRP) vaccine regimen by assessing protection against Turkey or Oman strains of CCHFV. We found that all mice were completely protected from disease, supporting broad applicability of this platform for CCHFV prevention.

Crimean-Congo hemorrhagic fever virus (CCHFV) infection can result in a severe hemorrhagic syndrome for which there are no antiviral interventions available to date. Certain RNA viruses, such as CCHFV, encode cysteine proteases of the ovarian tumor (OTU) family that antagonize interferon (IFN) production by deconjugating ubiquitin (Ub). The OTU of CCHFV, a negative-strand RNA virus, is dispensable for replication of the viral genome, despite being part of the large viral RNA polymerase. Here, we show that mutations that prevent binding of the OTU to cellular ubiquitin are required for the generation of recombinant CCHFV containing a mutated catalytic cysteine. Similarly, the high-affinity binding of a synthetic ubiquitin variant (UbV-CC4) to CCHFV OTU strongly inhibits viral growth. UbV-CC4 inhibits CCHFV infection even in the absence of intact IFN signaling, suggesting that its antiviral activity is not due to blocking the OTU's immunosuppressive function. Instead, the prolonged occupancy of the OTU with UbV-CC4 directly targets viral replication by interfering with CCHFV RNA synthesis. Together, our data provide mechanistic details supporting the development of antivirals targeting viral OTUs.IMPORTANCE Crimean-Congo hemorrhagic fever virus is an important human pathogen with a wide global distribution for which no therapeutic interventions are available. CCHFV encodes a cysteine protease belonging to the ovarian tumor (OTU) family which is involved in host immune suppression. Here we demonstrate that artificially prolonged binding of the OTU to a substrate inhibits virus infection. This provides novel insights into CCHFV OTU function during the viral replicative cycle and highlights the OTU as a potential antiviral target.

Lassa fever is a frequently severe human disease that is endemic to several countries in West Africa. To date, no licensed vaccines are available to prevent Lassa virus (LASV) infection, even though Lassa fever is thought to be an important disease contributing to mortality and both acute and chronic morbidity. We have previously described a vaccine candidate composed of single-cycle LASV replicon particles (VRPs) and a stable cell line for their production. Here, we refine the genetic composition of the VRPs and demonstrate the ability to reproducibly purify them with high yields. Studies in the guinea pig model confirm efficacy of the vaccine candidate, demonstrate that single-cycle replication is necessary for complete protection by the VRP vaccine, and show that post-exposure vaccination can confer protection from lethal outcome.

Crimean-Congo hemorrhagic fever virus (CCHFV) is an emerging tick-borne virus from the family Nairoviridae that frequently causes lethal disease in humans. CCHFV has a wide geographic distribution, and cases have been reported in Africa, Asia, the Middle East, and Europe. Recent autochthonous cases in Spain demonstrate the emergence of CCHFV in previously naïve regions [1]. CCHFV is considered a public health threat due to its epidemic potential, high case fatality rate, and lack of treatment options. Currently, no antivirals or licensed vaccines with proven efficacy are available. CCHFV was recently included on the World Health Organization Research and Development Blueprint list of infectious agents critically needing effective prophylaxis and therapeutics to prevent major outbreaks. Development of a safe and efficacious vaccine is critical for restricting future outbreaks and preventing disease in endemic countries. In an outbreak situation, a CCHFV vaccine should ideally confer maximal protection after administration of a single dose to rapidly protect at-risk populations.

Interferon-stimulated gene product 15 (ISG15) is a key component of host responses to microbial infection. Despite having been known for four decades, grasping the functions and features of ISG15 has been a slow and elusive process. Substantial work over the past two decades has greatly enhanced this understanding, revealing the complex and variable nature of this protein. This has unveiled multiple mechanisms of action that are only now beginning to be understood. In addition, it has uncovered diversity not only between how ISG15 affects different pathogens but also between the function and structure of ISG15 itself between different host species. Here we review the complexity of ISG15 within the context of viral infection, focusing primarily on its antiviral function and the mechanisms viruses employ to thwart its effects. We highlight what is known regarding the impact of ISG15 sequence and structural diversity on these interactions and discuss the aspects presenting the next frontier toward elucidating a more complete picture of ISG15 function.

Post-translational modification of host and viral proteins by ubiquitin (Ub) and Ub-like proteins, such as interferon stimulated gene product 15 (ISG15), plays a key role in response to infection. Viruses have been increasingly identified that contain proteases possessing deubiquitinase (DUB) and/or deISGylase functions. This includes viruses in the Nairoviridae family that encode a viral homologue of the ovarian tumor protease (vOTU). vOTU activity was recently demonstrated to be critical for replication of the often-fatal Crimean-Congo hemorrhagic fever virus, with DUB activity suppressing the type I interferon responses and deISGylase activity broadly removing ISG15 conjugated proteins. There are currently about 40 known nairoviruses classified into fourteen species. Recent genomic characterization has revealed a high degree of diversity, with vOTUs showing less than 25% amino acids identities within the family. Previous investigations have been limited to only a few closely related nairoviruses, leaving it unclear what impact this diversity has on vOTU function. To probe the effects of vOTU diversity on enzyme activity and specificity, we assessed representative vOTUs spanning the Nairoviridae family towards Ub and ISG15 fluorogenic substrates. This revealed great variation in enzymatic activity and specific substrate preferences. A subset of the vOTUs were further assayed against eight biologically relevant di-Ub substrates, uncovering both common trends and distinct preferences of poly-Ub linkages by vOTUs. Four novel X-ray crystal structures were obtained that provide a biochemical rationale for vOTU substrate preferences and elucidate structural features that distinguish the vOTUs, including a motif in the Hughes orthonairovirus species that has not been previously observed in OTU domains. Additionally, structure-informed mutagenesis provided the first direct evidence of a second site involved in di-Ub binding for vOTUs. These results provide new insight into nairovirus evolution and pathogenesis, and further enhances the development of tools for therapeutic purposes.