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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 vaccine against heterologous LASV challenge, demonstrating complete protection against clinical disease after a single vaccination dose.

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 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.

Objective:The coronavirus disease 2019 (COVID-19) pandemic has resulted in shortages of personal protective equipment (PPE), underscoring the urgent need for simple, efficient, and inexpensive methods to decontaminate masks and respirators exposed to severe acute respiratory coronavirus virus 2 (SARS-CoV-2). We hypothesized that methylene blue (MB) photochemical treatment, which has various clinical applications, could decontaminate PPE contaminated with coronavirus.Design:The 2 arms of the study included (1) PPE inoculation with coronaviruses followed by MB with light (MBL) decontamination treatment and (2) PPE treatment with MBL for 5 cycles of decontamination to determine maintenance of PPE performance.Methods:MBL treatment was used to inactivate coronaviruses on 3 N95 filtering facepiece respirator (FFR) and 2 medical mask models. We inoculated FFR and medical mask materials with 3 coronaviruses, including SARS-CoV-2, and we treated them with 10 µM MB and exposed them to 50,000 lux of white light or 12,500 lux of red light for 30 minutes. In parallel, integrity was assessed after 5 cycles of decontamination using multiple US and international test methods, and the process was compared with the FDA-authorized vaporized hydrogen peroxide plus ozone (VHP+O3) decontamination method.Results:Overall, MBL robustly and consistently inactivated all 3 coronaviruses with 99.8% to >99.9% virus inactivation across all FFRs and medical masks tested. FFR and medical mask integrity was maintained after 5 cycles of MBL treatment, whereas 1 FFR model failed after 5 cycles of VHP+O3.Conclusions:MBL treatment decontaminated respirators and masks by inactivating 3 tested coronaviruses without compromising integrity through 5 cycles of decontamination. MBL decontamination is effective, is low cost, and does not require specialized equipment, making it applicable in low- to high-resource settings.

In the absence of approved vaccines and therapeutics for use in humans, Nipah virus (NiV) 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.

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 postexposure vaccination can confer protection from lethal outcome.

BackgroundThe coronavirus disease 2019 (COVID-19) pandemic has resulted in severe shortages of personal protective equipment (PPE) necessary to protect front-line healthcare personnel. These shortages underscore the urgent need for simple, efficient, and inexpensive methods to decontaminate SARS-CoV-2-exposed PPE enabling safe reuse of masks and respirators. Efficient decontamination must be available not only in low-resourced settings, but also in well-resourced settings affected by PPE shortages. Methylene blue (MB) photochemical treatment, hitherto with many clinical applications including those used to inactivate virus in plasma, presents a novel approach for widely applicable PPE decontamination. Dry heat (DH) treatment is another potential low-cost decontamination method.MethodsMB and light (MBL) and DH treatments were used to inactivate coronavirus on respirator and mask material. We tested three N95 filtering facepiece respirators (FFRs), two medical masks (MMs), and one cloth community mask (CM). FFR/MM/CM materials were inoculated with SARS-CoV-2 (a Betacoronavirus), murine hepatitis virus (MHV) (a Betacoronavirus), or porcine respiratory coronavirus (PRCV) (an Alphacoronavirus), and treated with 10 µM MB followed by 50,000 lux of broad-spectrum light or 12,500 lux of red light for 30 minutes, or with 75°C DH for 60 minutes. In parallel, we tested respirator and mask integrity using several standard methods and compared to the FDA-authorized vaporized hydrogen peroxide plus ozone (VHP+O3) decontamination method. Intact FFRs/MMs/CM were subjected to five cycles of decontamination (5CD) to assess integrity using International Standardization Organization (ISO), American Society for Testing and Materials (ASTM) International, National Institute for Occupational Safety and Health (NIOSH), and Occupational Safety and Health Administration (OSHA) test methods.FindingsOverall, MBL robustly and consistently inactivated all three coronaviruses with at least a 4-log reduction. DH yielded similar results, with the exception of MHV, which was only reduced by 2-log after treatment. FFR/MM integrity was maintained for 5 cycles of MBL or DH treatment, whereas one FFR failed after 5 cycles of VHP+O3. Baseline performance for the CM was variable, but reduction of integrity was minimal.InterpretationMethylene blue with light and DH treatment decontaminated masks and respirators by inactivating three tested coronaviruses without compromising integrity through 5CD. MBL decontamination of masks is effective, low-cost and does not require specialized equipment, making it applicable in all-resource settings. These attractive features support the utilization and continued development of this novel PPE decontamination method.

Crimean-Congo hemorrhagic fever (CCHF) is the most widely distributed tick-borne viral infection in the world. Strikingly, reported mortality rates for CCHF are extremely variable, ranging from 5% to 80% (Whitehouse, 2004). CCHF virus (CCHFV, Nairoviridae) exhibits extensive genomic sequence diversity across strains (Deyde et al., 2006; Sherifi et al., 2014). It is currently unknown if genomic diversity is a factor contributing to variation in its pathogenicity. We obtained complete genome sequences of CCHFV directly from the tick reservoir. These new strains belong to a solitary lineage named Europe 2 that is circumstantially reputed to be less pathogenic than the epidemic strains from Europe 1 lineage. We identified a single tick-specific amino acid variant in the viral glycoprotein region that dramatically reduces its fusion activity in human cells, providing evidence that a glycoprotein precursor variant, present in ticks, has severely impaired function in human cells., eLife digest Crimean-Congo hemorrhagic fever (CCHF) is caused by infection with a virus spread by ticks in Europe, Africa and Asia. It can cause severe disease in humans, including high fevers and bleeding. How deadly CCHF is varies with between 5% to 80% of those infected dying. Scientists suspect genetic differences in various strains of the virus may account for the differences in death rates, but they do not know the exact mutations that make the CCHF virus more or less deadly. To learn more, scientists have sorted strains of CCHF virus into different groups based on how similar they are genetically. One group called Europe 2 infects many people in the Balkans, but it rarely causes illness. In fact, only two mild cases of illness have been associated with Europe 2 strains, while other CCHF virus strains circulating in this region have caused thousands of more severe illnesses. Now, Hua et al. identified a mutation in one Europe 2 strain of the CCHF virus that may explain why this subgroup of viruses rarely causes severe human disease. The researchers collected a strain of CCHF virus from infected ticks found in Bulgaria and sequenced its genome. They named the virus strain Malko Tarnovo. Through a series of experiments, Hua et al. showed that the Malko Tarnovo strain very efficiently infects tick cells but not human cells. A single amino acid change in the genetic sequence of the virus appears to make the virus less able to infect human cells. The mutation prevents a protein on the surface of the virus from fusing with human cells, an essential step in infection. This may explain why this strain and others in the Europe 2 group do not cause severe human disease. Hua et al. also demonstrate the importance of studying viruses in the animals that spread them. By studying the CCHF virus in ticks, scientists may be able to learn more about how viruses evolve to infect new species, which may help scientists prevent future pandemics.

The error-prone nature of 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). DI 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.

Antiviral responses are regulated by conjugation of ubiquitin (Ub) and interferon-stimulated gene 15 (ISG15) to proteins. Certain classes of viruses encode Ub- or ISG15-specific proteases belonging to the ovarian tumor (OTU) superfamily. Their activity is thought to suppress cellular immune responses, but studies demonstrating the function of viral OTU proteases during infection are lacking. Crimean-Congo hemorrhagic fever virus (CCHFV, family Nairoviridae) is a highly pathogenic human virus that encodes an OTU with both deubiquitinase and deISGylase activity as part of the viral RNA polymerase. We investigated CCHFV OTU function by inactivating protease catalytic activity or by selectively disrupting its deubiquitinase and deISGylase activity using reverse genetics. CCHFV OTU inactivation blocked viral replication independently of its RNA polymerase activity, while deubiquitinase activity proved critical for suppressing the interferon responses. Our findings provide insights into viral OTU functions and supports the development of therapeutics and vaccines.

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.

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.

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.

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., 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.

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,...

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., Graphical abstract Unlabelled Image, Highlights • ISG15 is one of the most highly upregulated IFN-stimulated proteins. • ISG15 is involved in numerous pathways with multiple mechanisms of action. • Viruses encode specific countermeasures to combat ISG15 function. • ISG15 possesses interspecies sequence diversity impacting structure, function, and viral interactions. • Key advances have been made; much work remains to grasp the complexity of ISG15.

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 (10200∆NSm) 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.

Genome reassortment in Lassa virus (LASV) has been reported in nature, but phenotypic consequences of this phenomenon are not well described. Here we characterize, both in vitro and in vivo, reassortment between 2 LASV strains: the prototypic 1976 Josiah strain and a more recently isolated 2015 Liberian strain. In vitro analysis showed that although cis- and trans-acting elements of viral RNA synthesis were compatible between strains, reassortants demonstrated different levels of viral replication. These differences were also apparent in vivo, as reassortants varied in pathogenicity in the guinea pig model of LASV infection. The reassortant variant containing the Josiah S segment retained the virulence of the parental Josiah strain, but the reassortant variant containing the S segment of the Liberian isolate was highly attenuated compared to both parental strains. Contrary to observations in reassortants between LASV and other arenavirus species, which suggest that L segment-encoded factors are responsible for virulence, these studies highlight a role for S segment-encoded virulence factors in disease, and also suggest that inefficient interactions between proteins of heterologous strains may limit the prevalence of reassortant LASV variants in nature.

Stress granules (SGs) are protein-mRNA aggregates that are formed in response to environmental stresses, resulting in translational inhibition. SGs are generally believed to play an antiviral role and are manipulated by many viruses, including various alphaviruses. GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1) is a key component and commonly used marker of SGs. Its homolog G3BP2 is a less extensively studied SG component. Here, we demonstrate that Chikungunya virus (CHIKV) infection induces cytoplasmic G3BP1- and G3BP2-containing granules that differ from bona fide SGs in terms of morphology, composition, and behavior. For several Old World alphaviruses it has been shown that nonstructural protein 3 (nsP3) interacts with G3BPs, presumably to inhibit SG formation, and we have confirmed this interaction in CHIKV-infected cells. Surprisingly, CHIKV also relied on G3BPs for efficient replication, as simultaneous depletion of G3BP1 and G3BP2 reduced viral RNA levels, CHIKV protein expression, and viral progeny titers. The G3BPs colocalized with CHIKV nsP2 and nsP3 in cytoplasmic foci, but no colocalization with nsP1, nsP4, or dsRNA was observed. Furthermore, G3BPs could not be detected in a cellular fraction enriched for CHIKV replication/transcription complexes, suggesting that they are not directly involved in CHIKV RNA synthesis. Depletion of G3BPs did not affect viral entry, translation of incoming genomes, or nonstructural polyprotein processing but resulted in severely reduced levels of negative-stranded (and consequently also positive-stranded) RNA. This suggests a role for the G3BPs in the switch from translation to genome amplification, although the exact mechanism by which they act remains to be explored. IMPORTANCE Chikungunya virus (CHIKV) causes a severe polyarthritis that has affected millions of people since its reemergence in 2004. The lack of approved vaccines or therapeutic options and the ongoing explosive outbreak in the Caribbean underline the importance of better understanding CHIKV replication. Stress granules (SGs) are cytoplasmic protein-mRNA aggregates formed in response to various stresses, including viral infection. The RNA-binding proteins G3BP1 and G3BP2 are essential SG components. SG formation and the resulting translational inhibition are generally considered an antiviral response, and many viruses manipulate or block this process. Late in infection, we and others have observed CHIKV nonstructural protein 3 in cytoplasmic G3BP1- and G3BP2-containing granules. These virally induced foci differed from true SGs and did not appear to represent replication complexes. Surprisingly, we found that G3BP1 and G3BP2 were also needed for efficient CHIKV replication, likely by facilitating the switch from translation to genome amplification early in infection.

Crimean-Congo hemorrhagic fever virus (CCHFV), a tick-borne orthonairovirus, causes a severe hemorrhagic disease in humans (Crimean-Congo hemorrhagic fever, CCHF). Currently, no vaccines are approved to prevent CCHF; treatment is limited to supportive care and the use of ribavirin, the therapeutic benefits of which remain unclear. CCHF is part of WHO’s priority list of infectious diseases warranting further research and development. To aid in the identification of new antiviral compounds, we generated a recombinant CCHFV expressing a reporter protein, allowing us to quantify virus inhibition by measuring the reduction in fluorescence in infected cells treated with candidate compounds. The screening assay was readily adaptable to high-throughput screening (HTS) of compounds using Huh7 cells, with a signal-to-noise ratio of 50:1, and Z′-factors > 0.6 in both 96- and 384-well formats. A screen of candidate nucleoside analog compounds identified 2′-deoxy-2′-fluorocytidine (EC(50) = 61 ± 18 nM) as having 200 × the potency of ribavirin (EC(50) = 12.5 ± 2.6 μM), as well as 17 × the potency of T-705 (favipiravir), another compound with reported anti-CCHFV activity (EC(50) = 1.03 ± 0.16 μM). Furthermore, we also determined that 2′-deoxy-2′-fluorocytidine acts synergistically with T-705 to inhibit CCHFV replication without causing cytotoxicity. The incorporation of this reporter virus into the high-throughput screening assay described here will allow more rapid identification of effective therapeutic options to combat this emerging human pathogen.

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus that causes severe persistent arthralgia. To better understand the molecular details of CHIKV RNA synthesis and the mode of action of inhibitors, we have developed an in vitro assay to study CHIKV replication/transcription complexes isolated from infected cells. In this assay 32P-CTP was incorporated into the CHIKV genome, subgenomic (sg) RNA and into a ~7.5 kb positive-stranded RNA, termed RNA II. We mapped RNA II, which was also found in CHIKV-infected cells, to the 5′ end of the genome up to the start of the sgRNA promoter region. Most of the RNA-synthesizing activity, negative-stranded RNA and a relatively large proportion of nsP1 and nsP4 were recovered from a crude membrane fraction obtained by pelleting at 15 000 . Positive-stranded RNA was mainly found in the cytosolic S15 fraction, suggesting it was released from the membrane-associated replication/transcription complexes (RTCs). The newly synthesized RNA was relatively stable and remained protected from cellular nucleases, possibly by encapsidation. A set of compounds that inhibit CHIKV replication in cell culture was tested in the in vitro RTC assay. In contrast to 3′dNTPs, chain terminators that acted as potent inhibitors of RTC activity, ribavirin triphosphate and 6-aza-UTP did not affect the RNA-synthesizing activity in vitro. In conclusion, this in vitro assay for CHIKV RNA synthesis is a useful tool for mechanistic studies on the RTC and mode of action studies on compounds with anti-CHIKV activity.

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-α/β 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., Author summary Human infection by tick-borne Crimean-Congo hemorrhagic fever virus (CCHFV) can result in severe disease with up to 30% case fatality rates. While CCHFV is known to be hepatotropic, the presence and implications of virus in other tissues are less clear. Furthermore, to date, early cellular targets of infection in a CCHFV disease model have not been investigated in detail. Here, using a recombinant reporter CCHFV expressing the fluorescent protein ZsGreen1 (ZsG; CCHFV/ZsG) in interferon-α/β receptor knock-out (Ifnar-/-) mice, which develop acute fatal disease following infection, we investigate both cellular and tissue targets of infection. Importantly, we find that CCHFV/ZsG infection demonstrated comparable pathogenicity to wild-type virus in Ifnar-/- mice. We used in situ visualization of fluorescent signal in tissues to assess viral dissemination throughout the course of infection, and found robust viral signal in reproductive tissues, previously unrecognized as sites of CCHFV infection. We also used flow cytometry to detect intracellular fluorescent signal, and identified initial target cells of CCHFV infection as macrophage and monocyte populations in lymphatic tissues. These findings support a central role of immune cells in early virus dissemination, and a need for further investigations into reproductive tract involvement in human CCHFV 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.

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., Author summary Viruses utilize a variety of mechanisms to manipulate and suppress host responses to infection. One specific mechanism used by nairoviruses is the production of a deubiquitinating enzyme, termed the vOTU, that disrupts the innate immune response. This enzyme has been shown to play a key role in efficient replication of Crimean-Congo hemorrhagic fever virus (CCHFV), a severe human pathogen causing outbreaks with high case fatality rates. Recent genomic studies have revealed a high degree of sequence variation for the vOTU among nairoviruses, but knowledge relating to the functional impact of this diversity is lacking. Here we investigated the effects of this diversity on the structure and function of vOTUs from a wide range of nairoviruses. This revealed that vOTUs from different nairoviruses possess distinct preferences for certain host proteins. In addition, we found that different vOTUs possess distinguishing structural features, including a unique motif present in one that was previously undescribed. Utilizing this information, we were able to provide a rational basis for the observed differences in the vOTUs. This work provides a foundation to understand nairovirus evolution by providing insight into a mechanism that influences virus host adaptation and pathogenesis.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne pathogen that causes high morbidity and mortality. Efficacy of vaccines and antivirals to treat human CCHFV infections remains limited and controversial. Research into pathology and underlying molecular mechanisms of CCHFV and other nairoviruses is limited. Significant progress has been made in our understanding of CCHFV replication and pathogenesis in the past decade. Here we review the most recent molecular advances in CCHFV-related research, and provide perspectives on future research.

The unfolded protein response (UPR) is a cellular defence mechanism against high concentrations of misfolded protein in the endoplasmic reticulum (ER). In the presence of misfolded proteins, ER-transmembrane proteins PERK and IRE1α become activated. PERK phosphorylates eIF2α leading to a general inhibition of cellular translation, whilst the expression of transcription factor ATF4 is upregulated. Active IRE1α splices out an intron from XBP1 mRNA, to produce a potent transcription factor. Activation of the UPR increases the production of several proteins involved in protein folding, degradation and apoptosis. Here, we demonstrated that transient expression of chikungunya virus (CHIKV) (family Togaviridae, genus Alphavirus) envelope glycoproteins induced the UPR and that CHIKV infection resulted in the phosphorylation of eIF2α and partial splicing of XBP1 mRNA. However, infection with CHIKV did not increase the expression of ATF4 and known UPR target genes (GRP78/BiP, GRP94 and CHOP). Moreover, nuclear XBP1 was not observed during CHIKV infection. Even upon stimulation with tunicamycin, the UPR was efficiently inhibited in CHIKV-infected cells. Individual expression of CHIKV non-structural proteins (nsPs) revealed that nsP2 alone was sufficient to inhibit the UPR. Mutations that rendered nsP2 unable to cause host-cell shut-off prevented nsP2-mediated inhibition of the UPR. This indicates that initial UPR induction takes place in the ER but that expression of functional UPR transcription factors and target genes is efficiently inhibited by CHIKV nsP2.

RIG-I is a cytosolic sensor critically involved in the activation of the innate immune response to RNA virus infection. In the present study, we evaluated the inhibitory effect of a RIG-I agonist on the replication of two emerging arthropod-borne viral pathogens, dengue virus (DENV) and chikungunya virus (CHIKV), for which no therapeutic options currently exist. We demonstrate that when a low, noncytotoxic dose of an optimized 5′triphosphorylated RNA (5′pppRNA) molecule was administered, RIG-I stimulation generated a robust antiviral response against these two viruses. Strikingly, 5′pppRNA treatment before or after challenge with DENV or CHIKV provided protection against infection. In primary human monocytes and monocyte-derived dendritic cells, the RIG-I agonist blocked both primary infection and antibody-dependent enhancement of DENV infection. The protective response against DENV and CHIKV induced by 5′pppRNA was dependent on an intact RIG-I/MAVS/TBK1/IRF3 axis and was largely independent of the type I IFN response. Altogether, this in vitro analysis of the antiviral efficacy of 5′pppRNA highlights the therapeutic potential of RIG-I agonists against emerging viruses such as DENV and CHIKV. IMPORTANCE DENV and CHIKV are two reemerging mosquito-borne viruses for which no therapeutic options currently exist. Both viruses overlap geographically in tropical regions of the world, produce similar fever-like symptoms, and are difficult to diagnose. This study investigated the inhibitory effect of a RIG-I agonist on the replication of these two viruses. RIG-I stimulation using 5′pppRNA before or after DENV or CHIKV infection generated a protective antiviral response against both pathogens in immune and nonimmune cells; interestingly, the protective response against the viruses was largely independent of the classical type I interferon response. The antiviral efficacy of 5′pppRNA highlights the therapeutic potential of RIG-I agonists against emerging viruses such as DENV and CHIKV.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that re-emerged in 2004 and has caused massive outbreaks in recent years. The lack of a licensed vaccine or treatment options emphasize the need to obtain more insight into the viral life cycle and CHIKV-host interactions. Infectious cDNA clones are important tools for such studies, and for mechanism of action studies on antiviral compounds. Existing CHIKV cDNA clones are based on a single genome from an individual clinical isolate, which is expected to have evolved specific characteristics in response to the host environment, and possibly also during subsequent cell culture passaging. To obtain a virus expected to have the general characteristics of the recent E1-226V CHIKV isolates, we have constructed a new CHIKV full-length cDNA clone, CHIKV LS3, based on the consensus sequence of their aligned genomes. Here we report the characterization of this synthetic virus and a green fluorescent protein-expressing variant (CHIKV LS3-GFP). Their characteristics were compared to those of natural strain ITA07-RA1, which was isolated during the 2007 outbreak in Italy. In cell culture the synthetic viruses displayed phenotypes comparable to the natural isolate, and in a mouse model they caused lethal infections that were indistinguishable from infections with a natural strain. Compared to ITA07-RA1 and clinical isolate NL10/152, the synthetic viruses displayed similar sensitivities to several antiviral compounds. 3-deaza-adenosine was identified as a new inhibitor of CHIKV replication. Cyclosporin A had no effect on CHIKV replication, suggesting that cyclophilins - opposite to what was found for other +RNA viruses- do not play an essential role in CHIKV replication. The characterization of the consensus sequence-based synthetic viruses and their comparison to natural isolates demonstrated that CHIKV LS3 and LS3-GFP are suitable and representative tools to study CHIKV-host interactions, screen for antiviral compounds and unravel their mode of action.