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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Chikungunya virus (CHIKV) is a reemerging alphavirus. Since 2005, it has infected millions of people during outbreaks in Africa, Asia, and South/Central America. CHIKV replication depends on host cell factors at many levels and is expected to have a profound effect on cellular physiology. To obtain more insight into host responses to infection, stable isotope labeling with amino acids in cell culture and liquid chromatography-tandem mass spectrometry were used to assess temporal changes in the cellular phosphoproteome during CHIKV infection. Among the ~3,000 unique phosphorylation sites analyzed, the largest change in phosphorylation status was measured on residue T56 of eukaryotic elongation factor 2 (eEF2), which showed a >50-fold increase at 8 and 12 h p.i. Infection with other alphaviruses (Semliki Forest, Sindbis and Venezuelan equine encephalitis virus (VEEV)) triggered a similarly strong eEF2 phosphorylation. Expression of a truncated form of CHIKV or VEEV nsP2, containing only the N-terminal and NTPase/helicase domains (nsP2-NTD-Hel), sufficed to induce eEF2 phosphorylation, which could be prevented by mutating key residues in the Walker A and B motifs of the NTPase domain. Alphavirus infection or expression of nsP2-NTD-Hel resulted in decreased cellular ATP levels and increased cAMP levels. This did not occur when catalytically inactive NTPase mutants were expressed. The wild-type nsP2-NTD-Hel inhibited cellular translation independent of the C-terminal nsP2 domain, which was previously implicated in directing the virus-induced host shut-off for Old World alphaviruses. We hypothesize that the alphavirus NTPase activates a cellular adenylyl cyclase resulting in increased cAMP levels, thus activating PKA and subsequently eukaryotic elongation factor 2 kinase. This in turn triggers eEF2 phosphorylation and translational inhibition. We conclude that the nsP2-driven increase of cAMP levels contributes to the alphavirus-induced shut-off of cellular protein synthesis that is shared between Old and New World alphaviruses. MS Data are available via ProteomeXchange with identifier PXD009381. Author summary: Chikungunya virus (CHIKV) is an important human pathogen from the alphavirus family that has caused outbreaks in Africa, Asia and the Americas. CHIKV replication strongly depends on resources of the host cell, which undergoes many changes during infection. Here, we have used phosphoproteomics to obtain more insight into such changes, which revealed that CHIKV infection induces phosphorylation of eukaryotic elongation factor 2 (eEF2). eEF2 regulates the elongation step of translation and its phosphorylation can strongly reduce protein synthesis. Previously, the nsP2 C-terminal domain was shown to reduce cellular transcription, which can prevent antiviral responses and promotes diversion of cellular resources to virus replication. Here, we describe that nsP2 also causes a shut-off of host-cell translation, which is mediated by the enzymatic activity of the nsP2 NTPase domain which acts through a previously unnoticed mechanism that involves cAMP, PKA and eEF2. Alphavirus infection or overexpression of nsP2 increased the cellular cAMP concentration, a second messenger that activates several signaling pathways, including the PKA and eEF2 kinases, of which the latter phosphorylates eEF2. Our observations highlight how alphaviruses modulate host cell metabolism on different levels and may account for poorly understood host shut-off effects observed in alphavirus-infected cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

To identify host factors relevant for severe acute respiratory syndrome-coronavirus (SARS-CoV) replication, we performed a small interfering RNA (siRNA) library screen targeting the human kinome. Protein kinases are key regulators of many cellular functions, and the systematic knockdown of their expression should provide a broad perspective on factors and pathways promoting or antagonizing coronavirus replication. In addition to 40 proteins that promote SARS-CoV replication, our study identified 90 factors exhibiting an antiviral effect. Pathway analysis grouped subsets of these factors in specific cellular processes, including the innate immune response and the metabolism of complex lipids, which appear to play a role in SARS-CoV infection. Several factors were selected for in-depth validation in follow-up experiments. In cells depleted for the β2 subunit of the coatomer protein complex (COPB2), the strongest proviral hit, we observed reduced SARS-CoV protein expression and a>2-log reduction in virus yield. Knockdown of the COPB2-related proteins COPB1 and Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1) also suggested that COPI-coated vesicles and/or the early secretory pathway are important for SARS-CoV replication. Depletion of the antiviral double-stranded RNA-activated protein kinase (PKR) enhanced virus replication in the primary screen, and validation experiments confirmed increased SARS-CoV protein expression and virus production upon PKR depletion. In addition, cyclin-dependent kinase 6 (CDK6) was identified as a novel antiviral host factor in SARSCoV replication. The inventory of pro- and antiviral host factors and pathways described here substantiates and expands our understanding of SARS-CoV replication and may contribute to the identification of novel targets for antiviral therapy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]