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Alphavirus infections cause multiple alterations in the intracellular environment that can have both positive and negative effects on viral replication. The Old World alphaviruses, such as Sindbis (SINV), chikungunya (CHIKV), and Semliki Forest viruses, hinder the ability of vertebrate cells to form stress granules (SGs). Previously, this inhibitory function was attributed to the hypervariable domain (HVD) of nsP3, which sequesters the key SG components, G3BP1 and G3BP2, and to the nsP3 macro domain. The macro domain possesses ADP-ribosylhydrolase activity, which can diminish the ADP-ribosylation of G3BP1 during viral replication. However, our recent findings do not support the prevailing notions. We demonstrate that the interactions between SINV- or CHIKV-specific nsP3s and G3BPs, and the ADP-ribosylhydrolase activity are not major contributors to the inhibitory process, at least when nsP3 is expressed at biologically relevant levels. Instead, the primary factors responsible for suppressing SG formation are virus-induced transcriptional and translational shutoffs that rapidly develop within the first few hours post infection. Poorly replicating SINV variants carrying mutated nsP3 HVD still inhibit SG development even in the presence of sodium arsenite (NaAs). Conversely, SINV mutants lacking transcription and/or translation inhibitory functions lose their ability to inhibit SG formation, despite expressing high levels of wt nsP3. Moreover, we found that stable cell lines expressing GFP-nsP3 fusions retain the capacity to form SGs when exposed to NaAs. However, our results do not rule out the possibility that additional alphavirus-induced changes in cell biology may contribute to the suppression of SG formation. [ABSTRACT FROM AUTHOR]

The prevalence and distribution of African alphaviruses such as chikungunya have increased in recent years. Therefore, a better understanding of the local distribution of alphaviruses in vectors across the African continent is important. Here, entomological surveillance was performed from 2014 to 2018 at selected sites in north-eastern parts of South Africa where alphaviruses have been identified during outbreaks in humans and animals in the past. Mosquitoes were collected using a net, CDC-light, and BG-traps. An alphavirus genus-specific nested RT-PCR was used for screening, and positive pools were confirmed by sequencing and phylogenetic analysis. We collected 64,603 mosquitoes from 11 genera, of which 39,035 females were tested. Overall, 1462 mosquito pools were tested, of which 21 were positive for alphaviruses. Sindbis (61.9%, N = 13) and Middelburg (28.6%, N = 6) viruses were the most prevalent. Ndumu virus was detected in two pools (9.5%, N = 2). No chikungunya positive pools were identified. Arboviral activity was concentrated in peri-urban, rural, and conservation areas. A range of Culicidae species, including Culex univittatus, Cx. pipiens s.l., Aedes durbanensis, and the Ae. dentatus group, were identified as potential vectors. These findings confirm the active circulation and distribution of alphaviruses in regions where human or animal infections were identified in South Africa. [ABSTRACT FROM AUTHOR]

Our laboratory has been developing a Sindbis viral (SV) vector platform for treatments of ovarian and other types of cancers. In this study we show that SV.IL-12 combined with an agonistic OX40 antibody can eliminate ovarian cancer in a Mouse Ovarian Surface Epithelial Cell Line (MOSEC) model and further prevent tumors in mice rechallenged with tumor cells after approximately 5 months. Treatment efficacy is shown to be dependent upon T-cells that are transcriptionally and metabolically reprogramed. An influx of immune cells to the tumor microenvironment occurs. Combination of sequences encoding both IL-12 and anti-OX40 into a single SV vector, SV.IgGOX40.IL-12, facilitates the local delivery of immunoregulatory agents to tumors enhancing the anti-tumor response. We promote SV.IgGOX40.IL-12 as a safe and effective therapy for multiple types of cancer. [ABSTRACT FROM AUTHOR]

Background: Wolbachia pipientis are bacterial endosymbionts of arthropods currently being implemented as biocontrol agents to reduce the global burden of arboviral diseases. Some strains of Wolbachia, when introduced into Aedes aegypti mosquitoes, reduce or block the replication of RNA viruses pathogenic to humans. The wAlbB strain of Wolbachia was originally isolated from Aedes albopictus, and when transinfected into Ae. aegypti, persists in mosquitoes under high temperature conditions longer than other strains. The utility of wAlbB to block a broad spectrum of RNA viruses has received limited attention. Here we test the ability of wAlbB to reduce or block the replication of a range of Flavivirus and Alphavirus species in cell culture. Methods: The C6/36 mosquito cell line was stably infected with the wAlbB strain using the shell-vial technique. The replication of dengue, West Nile and three strains of Zika (genus Flavivirus), and Ross River, Barmah Forest and Sindbis (genus Alphavirus) viruses was compared in wAlbB-infected cells with Wolbachia-free controls. Infectious virus titres were determined using either immunofocus or plaque assays. A general linear model was used to test for significant differences in replication between flaviviruses and alphaviruses. Results: Titres of all viruses were significantly reduced in cell cultures infected with wAlbB versus Wolbachia-free controls. The magnitude of reduction in virus yields varied among virus species and, within species, also among the strains utilized. Conclusion: Our results suggest that wAlbB infection of arthropods could be used to reduce transmission of a wide range of pathogenic RNA viruses. [ABSTRACT FROM AUTHOR]

The Togaviridae family comprises several New- and Old-World Alphaviruses that have been responsible for thousands of human illnesses, including the RNA arbovirus Chikungunya virus (CHIKV). Firstly, it was reported in Tanzania in 1952 but rapidly it spread to several countries from Europe, Asia, and the Americas. Since then, CHIKV has been circulating in diverse countries around the world, leading to increased morbidity rates. Currently, there are no FDA-approved drugs or licensed vaccines to specifically treat CHIKV infections. Thus, there is a lack of alternatives to fight against this viral disease, making it an unmet need. Structurally, CHIKV is composed of five structural proteins (E3, E2, E1, C, and 6k) and four non-structural proteins (nsP1-4), in which nsP2 represents an attractive antiviral target for designing novel inhibitors since it has an essential role in the virus replication and transcription. Herein, we used a rational drug design strategy to select some acrylamide derivatives to be synthesized and evaluated against CHIKV nsP2 and also screened on CHIKV-infected cells. Thus, two regions of modifications were considered for these types of inhibitors, based on a previous study of our group, generating 1560 possible inhibitors. Then, the 24 most promising ones were synthesized and screened by using a FRET-based enzymatic assay protocol targeting CHIKV nsP2, identifying LQM330 , 333 , 336 , and 338 as the most potent inhibitors, with K i values of 48.6 ± 2.8, 92.3 ± 1.4, 2.3 ± 1.5, and 181.8 ± 2.5 μM, respectively. Still, their K m and V max kinetic parameters were also determined, along with their competitive binding modes of CHIKV nsP2 inhibition. Then, ITC analyses revealed K D values of 127, 159, 198, and 218 μM for LQM330 , 333 , 336 , and 338 , respectively. Also, their Δ H , Δ S , and Δ G physicochemical parameters were determined. MD simulations demonstrated that these inhibitors present a stable binding mode with nsP2, interacting with important residues of this protease, according to docking analyzes. Moreover, MM/PBSA calculations displayed that van der Waals interactions are mainly responsible for stabilizing the inhibitor-nsP2 complex, and their binding energies corroborated with their K i values, having −198.7 ± 15.68, −124.8 ± 17.27, −247.4 ± 23.78, and −100.6 ± 19.21 kcal/mol for LQM330 , 333 , 336 , and 338 , respectively. Since Sindbis (SINV) nsP2 is similar to CHIKV nsP2, these best inhibitors were screened against SINV-infected cells, and it was verified that LQM330 presented the best result, with an EC 50 value of 0.95 ± 0.09 μM. Even at 50 μM concentration, LQM338 was found to be cytotoxic on Vero cells after 48 h. Then, LQM330 , 333 , and 336 were evaluated against CHIKV-infected cells in antiviral assays, in which LQM330 was found to be the most promising antiviral candidate in this study, exhibiting an EC 50 value of 5.2 ± 0.52 μM and SI of 31.78. The intracellular flow cytometry demonstrated that LQM330 is able to reduce the CHIKV cytopathogenic effect on cells, and also reduce the percentage of CHIKV-positive cells from 66.1% ± 7.05 to 35.8% ± 5.78 at 50 μM concentration. Finally, qPCR studies demonstrated that LQM330 was capable of reducing the number of viral RNA copies/μL, suggesting that CHIKV nsP2 is targeted by this inhibitor as its mechanism of action. [Display omitted] • Four acrylamides were developed as competitive CHIKV nsP2 inhibitors. • ITC analyzes provided their Δ H , Δ S , Δ G , and also K D parameters. • MD simulations and MM/PBSA revealed main intermolecular forces and interactions. • LMQ330 was found to be the best antiviral against SINV- and CHIKV-infected cells. • qPCR demonstrated that LQM330 reduces the number of CHIKV vRNA copies. [ABSTRACT FROM AUTHOR]

Inherent in the study of viruses is the risk of pathogenic exposure, which necessitates appropriate levels of biosafety containment. Unfortunately, this also limits the availability of useful research instruments that are located at facilities not equipped to handle infectious pathogens. Abrogation of viral infectivity can be accomplished without severely disrupting the physical structure of the virus particle. Virus samples that are verifiably intact but not infectious may be enabled for study at research facilities where they would otherwise not be allowed. Inactivated viruses are also used in the development of vaccines, where immunogenicity is sought in the absence of active infection. We demonstrate the inactivation of Sindbis alphavirus particles in solution, as well as in crystallized form. Inactivation was accomplished by two different approaches: crosslinking of proteins by glutaraldehyde treatment, and crosslinking of nucleic acids by UV irradiation. Biophysical characterization methods, including dynamic light scattering and transmission electron microscopy, were used to demonstrate that the glutaraldehyde and UV inactivated Sindbis virus particles remain intact structurally. SDS-PAGE was also used to show evidence of the protein crosslinking that was expected with glutaraldehyde treatment, but also observed with UV irradiation. [ABSTRACT FROM AUTHOR]

Introduction: Mosquito-borne viruses have a widespread distribution across the globe and are known to pose serious threats to human and animal health. The maintenance and dissemination of these viruses in nature are driven through horizontal and vertical transmission. In the temperate climate of northern Sweden, there is a dearth of knowledge on whether mosquito-borne viruses that occur are transmitted transovarially. To gain a better understanding of mosquito-borne virus circulation and maintenance, mosquito larvae were sampled in northern Sweden during the first and second year after a large outbreak of Ockelbo disease in 2013 caused by Sindbis virus (SINV). Materials and Methods: A total of 3123 larvae were sampled during the summers of 2014 and 2015 at multiple sites in northern Sweden. The larvae were homogenized and screened for viruses using RT-PCR and sequencing. Species identification of selected larvae was performed by genetic barcoding targeting the cytochrome C oxidase subunit I gene. Results and Discussion: SINV RNA was detected in mosquito larvae of three different species, Ochlerotatus (Oc.) communis, Oc. punctor, and Oc. diantaeus. Inkoo virus (INKV) RNA was detected in Oc. communis larvae. This finding suggested that these mosquitoes could support transovarial transmission of SINV and INKV. Detection of virus in mosquito larva may serve as an early warning for emerging arboviral diseases and add information to epidemiological investigations before, during, and after outbreaks. Furthermore, our results demonstrated the relevance of genetic barcoding as an attractive and effective method for mosquito larva typing. However, further mosquito transmission studies are needed to ascertain the possible role of different mosquito species and developmental stages in the transmission cycle of arboviruses. [ABSTRACT FROM AUTHOR]

Alphaviruses cause debilitating disease in humans and animals and are transmitted by blood-feeding arthropods, typically mosquitoes. With a traditional focus on two models, Sindbis virus and Semliki Forest virus, alphavirus research has significantly intensified in the last decade partly due to the re-emergence and dramatic expansion of chikungunya virus in Asia, Europe, and the Americas. As a consequence, alphavirus-host interactions are now understood in much more molecular detail, and important novel mechanisms have been elucidated. It has become clear that alphaviruses not only cause a general host shut-off in infected vertebrate cells, but also specifically suppress different host antiviral pathways using their viral nonstructural proteins, nsP2 and nsP3. Here we review the current state of the art of alphavirus host cell shut-off of viral transcription and translation, and describe recent insights in viral subversion of interferon induction and signaling, the unfolded protein response, and stress granule assembly. [ABSTRACT FROM AUTHOR]

Alphaviruses are small, enveloped RNA viruses that form infectious particles by budding through the cellular plasma membrane. To help visualize and understand the intracellular assembly of alphavirus virions we have developed a bimolecular fluorescence complementation-based system (BiFC) that allows visualization of capsid and E2 subcellular localization and association in live cells. In this system, N- or C-terminal Venus fluorescent protein fragments (VN- and VC-) are fused to the N-terminus of the capsid protein on the Sindbis virus structural polyprotein, which results in the formation of fluorescent capsid-like structures in the absence of viral genomes that associate with the plasma membrane of cells. Mutation of the capsid autoprotease active site blocks structural polyprotein processing and alters the subcellular distribution of capsid fluorescence. Incorporating mCherry into the extracellular domain of the E2 glycoprotein allows the visualization of E2 glycoprotein localization and showed a close association of the E2 and capsid proteins at the plasma membrane as expected. These results suggest that this system is a useful new tool to study alphavirus assembly in live cells and may be useful in identifying molecules that inhibit alphavirus virion formation. [ABSTRACT FROM AUTHOR]

Mosquito-borne alphaviruses have the potential to cause large outbreaks throughout the world. Here we investigated the causative agent of an unexpected Sindbis virus (SINV) outbreak during August-September, 2013, in a previously nonendemic region of Sweden. Mosquitoes were collected using carbon dioxide-baited CDC traps at locations close to human cases. The mosquitoes were initially screened as large pools by SINV-specific quantitative RT-PCR, and the SINV-positive mosquitoes were species determined by single-nucleotide polymorphism (SNP) analysis, followed by sequencing the barcoding region of the cytochrome oxidase I gene. The proportion of the collected mosquitoes was determined by a metabarcoding strategy. By using novel strategies for PCR screening and genetic typing, a new SINV strain, Lövånger, was isolated from a pool of 1600 mosquitoes composed of Culex, Culiseta, and Aedes mosquitoes as determined by metabarcoding. The SINV-positive mosquito Culiseta morsitans was identified by SNP analysis and sequencing. After whole-genome sequencing and phylogenetic analysis, the SINV Lövånger isolate was shown to be most closely similar to recent Finnish SINV isolates. In conclusion, within a few weeks, we were able to detect and isolate a novel SINV strain and identify the mosquito vector during a sudden SINV outbreak. [ABSTRACT FROM AUTHOR]

Two species of arbovirus vector, Culex torrentium and Culex pipiens (Diptera: Culicidae), occur in several European countries, but difficulties in their accurate identification and discrimination have hampered both detailed and large-scale distribution and abundance studies. Using a molecular identification method, we identified to species 2559 larvae of Cx. pipiens/torrentium collected from 138 sites in 13 European countries ranging from Scandinavia to the Mediterranean coast. In addition, samples of 1712 males of Cx. pipiens/torrentium collected at several sites in the Czech Republic were identified to species based on the morphology of their hypopygia. We found that the two species occur together in large areas of Europe, and that Cx. torrentium dominates in northern Europe and Cx. pipiens dominates south of the Alps. The transition in dominance occurs in central Europe, where both species are roughly equally common. There was a strong correlation between the length of the growing season at different sites and occurrences of the two species. As the growing season increases, the proportion and detection of Cx. torrentium decrease, whereas those of Cx. pipiens increase. The present findings have important consequences for the interpretation of the results of studies on major enzootic and link-vectors of mosquito-borne bird-associated viruses (i.e. Sindbis, West Nile and Usutu viruses), especially in central Europe and Scandinavia. [ABSTRACT FROM AUTHOR]

Vaccinia virus protein A33 (A33VACV) plays an important role in protection against orthopoxviruses, and hence is included in experimental multi-subunit smallpox vaccines. In this study we show that single-dose vaccination with recombinant Sindbis virus expressing A33VACV, is sufficient to protect mice against lethal challenge with vaccinia virus WR (VACV-WR) and ectromelia virus (ECTV) but not against cowpox virus (CPXV), a closely related orthopoxvirus. Moreover, a subunit vaccine based on the cowpox virus A33 ortholog (A33CPXV) failed to protect against cowpox and only partially protected mice against VACV-WR challenge. We mapped regions of sequence variation between A33VACV and A33CPXVand analyzed the role of such variations in protection. We identified a single protective region located between residues 104-120 that harbors a putative H-2Kd T cell epitope as well as a B cell epitope - a target for the neutralizing antibody MAb-1G10 that blocks spreading of extracellular virions. Both epitopes in A33CPXV are mutated and predicted to be non-functional. Whereas vaccination with A33VACV did not induce in-vivo CTL activity to the predicted epitope, inhibition of virus spread in-vitro, and protection from lethal VACV challenge pointed to the B cell epitope highlighting the critical role of residue L118 and of adjacent compensatory residues in protection. This epitope's critical role in protection, as well as its modifications within the orthopoxvirus genus should be taken in context with the failure of A33 to protect against CPXV as demonstrated here. These findings should be considered when developing new subunit vaccines and monoclonal antibody based therapeutics against orthopoxviruses, especially variola virus, the etiologic agent of smallpox. [ABSTRACT FROM AUTHOR]

Highlights: [•] Sindbis-VLP replicon vectors expressing HIV Env induce CTL and prime for antibodies. [•] Replicon activity is sufficient to induce immunity. [•] Env-incorporation within VLP may contribute to antibody priming. [•] Antibody responses generated appear to be longer lived than those induced by protein. [ABSTRACT FROM AUTHOR]

Abstract: The AR86 strain of Sindbis virus causes lethal neurologic disease in adult mice. Previous studies have identified a virulence determinant at nonstructural protein (nsP) 1 position 538 that regulates neurovirulence, modulates clearance from the CNS, and interferes with the type I interferon pathway. The studies herein demonstrate that in the absence of type I interferon signaling, the attenuated mutant exhibited equivalent virulence to S300 virus. Furthermore, both S300 and nsP1 T538I viruses displayed similar neurovirulence and replication kinetics in IPS-1−/− mice. TRIF dependent signaling played a modest role in protecting against disease by both S300 and nsP1 T538I, but did not contribute to control of nsP1 T538I replication within the CNS, while MyD88 played no role in the disease process. These results indicate that the control of the nsP1 T538I mutant virus is largely mediated by IPS-1-dependent RLR signaling, with TRIF-dependent TLR signaling also contributing to protection from virus-induced neurologic disease. [Copyright &y& Elsevier]

Abstract: Alphaviruses are enveloped, single-stranded positive sense RNA viruses that are transmitted by an arthropod vector to a wide host range, including avian and mammalian species. Arthropods and vertebrates have different cellular environments and this may cause the different cellular pathologies that are observed between the invertebrate vector and vertebrate hosts in both whole organisms and cultured cell lines. In this report, we used Sindbis virus and examined mosquito and mammalian cell lines for their ability to produce progeny virus particles. Total particles produced, viral titers, and overall infectivity (or the ratio of total particles-to-infectious particles) was investigated. Our results show (1) Sindbis infectivity is more a function of the host cell used in titering the virus rather than the cell line used to produce the virus, (2) the number of total and infectious particles produced is cell line dependent, and (3) the infectivity of released virus particles improves during the course of infection in both cells that have cytolytic infections and persistent infections. [Copyright &y& Elsevier]

Abstract: Sindbis virus is a prototypic member of the Alphavirus genus, Togaviridae family. Sindbis replication results in cellular cytotoxicity, a feature that has been exploited by our laboratory for treatment of in vivo tumors. Understanding the interactions between Sindbis vectors and the host cell can lead to better virus production and increased efficacy of gene therapy vectors. Here we present studies investigating a possible cellular response to genotoxic effects of Sindbis vector infection. The Ataxia Telangiectasia Mutated (ATM) kinase, a sentinel against genomic and cellular stress, was activated by Sindbis vector infection at 3h post infection. ATM substrates, Mcm3 and the γH2AX histone, were subsequently phosphorylated, however, substrates involved with checkpoint arrest of DNA replication, p53, Chk1 and Chk2, were not differentially phosphorylated compared with uninfected cells. The ATM response suggests nuclear pertubation, resulting from cessation of host protein synthesis, as an early event in Sindbis vector infection. [Copyright &y& Elsevier]

Abstract: Sindbis virus (SINV) induces inflammatory and vasoactive responses that are associated with rash and arthritis in human infections. The mechanisms underlying infection-associated microvasculopathy are still unknown. We investigated whether endothelial cells infected by SINV are differentially responsive to bradykinin (BK), a potent inducer of inflammatory edema in a broad range of infectious diseases. Human endothelial cells (HBMECs) infected with SINV presented an upregulation of bradykinin B2 receptors (BK2R) expression. Also, BK reduced SINV-induced apoptosis and enhanced virus replication in HBMECs in a way dependent on BK2R, PI3 kinase and ERK signaling. Strikingly, intracerebral infection of mice in the presence of a BK2R antagonist reduced the local viral load. Our data suggest that SINV infection renders human endothelial cells hypersensitive to BK, which increases host cell survival and viral replication. Ongoing studies may clarify if the deregulation of the kinin pathway contributes to infection-associated vasculopathies in life-threatening arbovirus infections. [Copyright &y& Elsevier]

Abstract: Genetically engineered Sindbis viruses (SIN) are excellent oncolytic agents in preclinical models. Several human cancers have aberrant Akt signaling, and kinase inhibitors including rapamycin are currently tested in combination therapies with oncolytic viruses. Therefore, it was of interest to delineate possible cross-regulation between SIN replication and PI3K/Akt/mTOR signaling. Here, using HEK293T cells as host, we report the following key findings: (a) robust SIN replication occurs in the presence of mTOR specific inhibitors, rapamycin and torin1 or Ly294002 – a PI3K inhibitor, suggesting a lack of requirement for PI3K/Akt/mTOR signaling; (b) suppression of phosphorylation of Akt, mTOR and its effectors S6, and 4E-BP1 occurs late during SIN infection: a viral function that may be beneficial in counteracting cellular drug resistance to kinase inhibitors; (c) Ly294002 and SIN act additively to suppress PI3K/Akt/mTOR pathway with little effect on virus release; and (d) SIN replication induces host translational shut off, phosphorylation of eIF2α and apoptosis. This first report on the potent inhibition of Akt/mTOR signaling by SIN replication, bolsters further studies on the development and evaluation of engineered SIN genotypes in vitro and in vivo for unique cytolytic functions. [Copyright &y& Elsevier]

Abstract: Alphavirus persistence in the insect vector is an essential element in the vector–host transmission cycle of the virus and provides a model to study the biochemical and molecular basis for virus–vector coexistence. The prototype alphavirus Sindbis (SV) establishes persistent infections in invertebrate cell cultures which are characterized by low levels of virus production. We hypothesized that antiviral factors may be involved in decreasing the virus levels as virus persistence is established in invertebrate cells. Transcription profiles in Drosophila S2 cells at 5 days post-infection with SV identified families of gene products that code for factors that can explain previous observations seen in insect cells infected with alphaviruses. Genomic array analysis identified up-regulation of gene products involved in intracellular membrane vesicle formation, cell growth rate changes and immune-related functions in S2 cells infected with SV. Transcripts coding for factors involved in different aspects of the Notch signaling pathway had increased in expression. Increased expression of ankyrin, plap, syx13, unc-13, csp, rab1 and rab8 may aid in formation of virus containing vesicles and in intracellular transport of viral structural proteins. Possible functions of these gene products and relevant hypotheses are discussed. We confirmed the up-regulation of a wide-spectrum protease inhibitor, Thiol-ester containing Protein (TEP) II. We report inhibition of the viral polyprotein cleavage at 5 days post-infection (dpi) and after superinfection of SV-infected cells at 5dpi. We propose that inefficient cleavage of the polyprotein may, at least in part, lead to reduced levels of virus seen as persistence is established. [Copyright &y& Elsevier]

Infection of cultured cells with lytic animal viruses often results in the selective inhibition of host protein synthesis, whereas viral mRNA is efficiently translated under these circumstances. This phenomenon, known as "shut off," has been well described at the molecular level for some viruses, but there is not yet any direct or indirect evidence supporting the idea that it also should operate in animals infected with viruses. To address this issue, we constructed recombinant. Sindbis virus (SV)-expressing reporter mRNA, the translation of which is sensitive or resistant to virus-induced shut off. As found in cultured cells, replication of SV in mouse brain was associated with a strong phosphorylation of eukaryotic initiation factor (elF2) that prevented translation of reporter mRNA (luciferase and EGFP). Translation of these reporters was restored in vitro, in vivo, and ex vivo when a viral RNA structure, termed downstream hairpin loop, present in viral 26S mRNA, was placed at the 5' end of reporter mRNAs. By comparing the expression of shut off-sensitive and -resistant reporters, we unequivocally concluded that replication of SV in animal tissues is associated with a profound inhibition of nonviral mRNA translation. A strategy as simple as that followed here might be applicable to other viruses to evaluate their interference on host translation in infected animals. [ABSTRACT FROM AUTHOR]

Abstract: Alphaviruses are mosquito-borne viruses that cause serious human and animal diseases. Previous studies demonstrated that a determinant within the nsP1/nsP2 cleavage domain of the virulent Sindbis AR86 virus played a key role in regulating adult mouse virulence without adversely affecting viral replication. Additional characterization of this determinant demonstrated that a virus with the attenuating mutation induced more type I IFN production both in vivo and in vitro. Interestingly, this phenotype was not specific to the Sindbis AR86 virus, as a similar mutation in a distantly related alphavirus, Ross River Virus (RRV), also led to enhanced IFN induction. This effect was independent of virus-induced host shutoff, since IRF-3 phosphorylation, which occurs independently of de novo host transcription/translation, was induced more robustly in cells infected with the mutant viruses. Altogether, these results demonstrate that critical determinants within the nsP1/nsP2 cleavage domain play an important role in regulating alphavirus-induced IFN responses. [Copyright &y& Elsevier]

Whataroa virus, first detected in 1962 in bird populations around Whataroa township, is the only mosquito-borne virus circulating in New Zealand that has been isolated. Even though at least one other mosquito-borne pathogen (avian malaria) has greatly increased in prevalence in New Zealand in recent decades, no surveillance for Whataroa virus in its vertebrate hosts has been carried out for four decades. This is of concern since Whataroa virus may infect humans, possibly causing influenza-like symptoms. Here we reassess the status of Whataroa virus in the same bird species in the same area where it was detected previously. Molecular diagnostics identified Whataroa virus in three out of 95 non-native birds screened: two out of eight song thrushes Turdus philomelos, and one out of nine blackbirds Turdus merula. The detection of virus in birds, in contrast with recent screening of mosquitoes, highlights how wildlife surveillance for pathogens can be far more effective than vector surveillance. Results of this survey indicate the virus has not increased substantially in prevalence since last monitored, possibly because of little change in the local mosquito vector community. Finally, virus detection in the two Turdus species alone supports earlier claims that these non-native hosts act as reservoirs that maintain the virus, sourcing spillover infections in other native and non-native species. A similar role for the blackbird in avian malaria epidemiology in New Zealand has also recently been hypothesized. [ABSTRACT FROM AUTHOR]

Abstract: A self-replicating RNA vaccine encoding rabies virus glycoprotein gene was developed utilizing sindbis virus RNA replicon. The in vitro transcribed RNA (Sin-Rab-G RNA) was transfected in mammalian cells and analysed for self-replication and expression of rabies glycoprotein. To generate immune responses against rabies, mice were immunized with 10μg of Sin-Rab-G RNA and immune responses developed were compared with mice immunized with rabies DNA vaccine and commercial cell culture vaccine (Rabipur). The self-replicating rabies RNA vaccine generated cellular and humoral IgG responses similar to rabies DNA vaccine. On challenge with rabies virus CVS strain, rabies RNA vaccine conferred protection similar to rabies DNA vaccine. These results demonstrated that replicon-based self-replicating rabies RNA vaccine with 10μg dose was effective in inducing immune responses and protection similar to rabies DNA vaccine. [Copyright &y& Elsevier]

Mosquito-borne viruses cause significant levels of morbidity and mortality in humans and domesticated animals. Maintenance of mosquito-borne viruses in nature requires a biological transmission cycle that involves alternating virus replication in a susceptible vertebrate and mosquito host. Although the vertebrate infection is acute and often associated with disease, continual transmission of these viruses in nature depends on the establishment of a persistent, nonpathogenic infection in the mosquito vector. An antiviral RNAi response has been shown to limit the replication of RNA viruses in flies. However, the importance of the RNAi pathway as an antiviral defense in mammals is unclear. Differences in the immune responses of mammals and mosquitoes may explain why these viruses are not generally associated with pathology in the invertebrate host. We identified virus-derived small interfering RNAs (viRNAs), 21 nt in length, in Aedes aegypti infected with the mosquito-borne virus, Sindbis (SINV). viRNAs had an asymmetric distribution that spanned the length of the SINV genome. To determine the role of viRNAs in controlling pathogenic potential, mosquitoes were infected with recombinant alphaviruses expressing suppressors of RNA silencing. Decreased survival was observed in mosquitoes in which the accumulation of viRNAs was suppressed. These results suggest that an exogenous siRNA pathway is essential to the survival of mosquitoes infected with alphaviruses and, thus, the maintenance of these viruses in nature. [ABSTRACT FROM AUTHOR]

Abstract: A sindbis virus replicon-based DNA vaccine encoding rabies virus glycoprotein (G) was developed by subcloning rabies G gene into a sindbis virus replicon-based vaccine vector (pAlpha). The self-amplification of RNA transcripts and translation efficiency of rabies G was analyzed in pAlpha-Rab-G-transfected mammalian cells using RT-PCR, SDS-PAGE and Western blot analysis. The transfected cells also showed induction of apoptosis which is an important event in the enhancement of immune responses. Further, immune responses induced with replicon-based rabies DNA vaccine (pAlpha-Rab-G) was compared with conventional rabies DNA vaccine and commercial cell culture vaccine (Rabipur) in intramuscularly injected mice. The mice immunized with replicon-based rabies DNA vaccine induced humoral and cell mediated immune responses better than conventional rabies DNA vaccine however, comparable to Rabipur vaccine. On challenge with rabies virus CVS strain, replicon-based rabies DNA vaccine conferred complete protection similar to Rabipur. These results demonstrate that replicon-based rabies DNA vaccine is effective in inducing both humoral and cellular immune responses and can be considered as effective vaccine against rabies. [Copyright &y& Elsevier]

Aims: To compare the inactivation rate of Venezuelan equine encephalomyelitis (VEE) virus in liquids to that of Sindbis virus (SV, another alphavirus) and to a bacteriophage (MS2) generally used as a viral simulant in the development of countermeasures in biodefense. Methods and Results: Viruses were inoculated into liquids and viral titres were determined at various times postinoculation. The viruses were stable in distilled-deionized (dd) water at 4°C during the 21 days of the study. The inactivation rates of VEE and SV in dd water at 21 and 30°C were very similar (between 0·12 and 0·14 log10 per day), while MS2 was three-fold slower. In tap water (chlorine content between 4 and 5 ppm) at 21°C, VEE and SV were inactivated at twice the rate measured in dd water. Conclusions: The inactivation rates of VEE and SV were similar to each other and faster than MS2 in all liquids tested. Significance and Impact of the Study: VEE is likely to remain viable for many days after release into water, snow, or even chlorinated tap water. SV can be used to estimate the persistence of VEE in liquids, but using MS2 as a simulant would overestimate of the stability of VEE. [ABSTRACT FROM AUTHOR]

Abstract: We report here on the use of a Sindbis virus-based DNA-launch RNA replicon vector (pSIN-HA) that expresses influenza hemagglutinin (HA) as an immunogen. Immunization of mice with pSIN-HA generated anti-HA antibody and CTL responses and resulted in lower lung viral titers after influenza challenge when compared to controls. Importantly, immunization with a low dose of pSIN-HA mediated significantly reduced lung viral titers following challenge at 43 weeks after the final immunization. In contrast, immunization with a non-replicon DNA vector expressing HA failed to mediate reduced lung viral titer at the same dose. This demonstrated the dose-sparing capacity of the SIN vector system and its ability to stimulate long-term memory responses, properties that are highly desirable in any vaccine formulation. [Copyright &y& Elsevier]

Abstract: The role of interferon-gamma (IFNγ) in antiviral innate immune responses during acute alphavirus infection is not well defined. We examined the contribution of IFNγ to the protection of adult mice from Sindbis virus (SB)-induced disease by comparing subcutaneous infection of mice lacking receptors for either IFNα/β (A129), IFNγ (G129) or both (AG129) to normal mice (WT129). While neither G129 nor WT129 mice exhibited clinical signs of disease, infection of A129 or AG129 mice was fatal with AG129 mice succumbing more rapidly. Furthermore, AG129 mice developed signs of viral hemorrhagic fever (VHF), including extensive hepatocellular damage, inflammatory infiltrates in multiple organs and vascular leakage, which were significantly delayed and/or partially ameliorated during fatal A129 infections. We conclude that: (i) IFNα/β is the primary mediator of innate immunity to SB infection, however; (ii) IFNγ is directly antiviral in vivo, acting before the adaptive immune response appears and; (iii) development of VHF may involve viral suppression of both IFNα/β and IFNγ responses. [Copyright &y& Elsevier]

Introduction : Small rodents inhabiting the wadis close to St Katherine in the mountains of the Sinai Peninsula, Egypt, were trapped and antibodies to Sindbis (SIN) , West Nile (WN) and Quaranfil (QRF) viruses in their sera were studied. Materials and Methods One mouse had Haemagglutination-inhibition (HI) antibodies against SIN antigen in his serum, 3 mice had HI antibodies against WN antigen in their sera. The titers is low to be of any significance. Conclusion: It seems that natural cycle of transmission of SIN and WN viruses involving rodents does not exist. Seven mice had Complement-fixation (CF) antibodies in their sera against Quaranfil antigen. Conclusion, the CF test results suggest the existence of another mammal vector cycle. [ABSTRACT FROM AUTHOR]

Abstract: Sindbis is an Alphavirus capable of infecting and replicating in both vertebrate and invertebrate hosts. Mature Sindbis virus particles consist of an inner capsid surrounded by a host-derived lipid bilayer, which in turn is surrounded by a protein shell consisting of the E1 and E2 glycoproteins. While a homolog of the E1 glycoprotein has been structurally characterized, the amount of structural data on the E2 glycoprotein is considerably less. In this study, the organization of the E2 glycoprotein was probed by surface biotinylation of intact virions. The virus remained fully infectious, demonstrating that the biotinylation did not alter the topology of the proteins involved in infection. Seven sites of modification were identified in the E2 glycoprotein (K70, K76, K97, K131, K149, K202, and K235), while one site of modification in the E1 glycoprotein (K16) was identified, confirming that the E1 protein is almost completely buried in the virus structure. [Copyright &y& Elsevier]

Abstract: Some viruses display enhanced infection for Fc receptor (FcR)-positive cell types when complexed with virus-specific immunoglobulin (Ig). This process has been termed antibody-dependent enhancement of viral infection (ADE). We reasoned that the mechanism of ADE could be exploited and adapted to target alphavirus-based vectors to FcR-positive cell types. Towards this goal, recombinant Sindbis viruses were constructed that express 1 to 4 immunoglobulin-binding domains of protein L (PpL) as N-terminal extensions of the E2 glycoprotein. PpL is a bacterial protein that binds the variable region of antibody kappa light chains from a range of mammalian species. The recombinant viruses incorporated PpL/E2 fusion proteins into the virion structure and recapitulated the species-specific Ig-binding phenotypes of native PpL. Virions reacted with non-immune serum or purified IgG displayed enhanced binding and ADE for several species-matched FcR-positive murine and human cell lines. ADE required virus expression of a functional PpL Ig-binding domain, and appeared to be FcγR-mediated. Specifically, ADE did not occur with FcγR-negative cells, did not require active complement proteins, and did not occur on FcγR-positive murine cell lines when virions were bound by murine IgG-derived F(ab′)2 fragments. [Copyright &y& Elsevier]

Abstract: Sindbis virus particles are composed of three structural proteins (Capsid/E2/E1). In the mature virion the E1 glycoprotein is organized in a highly constrained, energy-rich conformation. It is hypothesized that this energy is utilized to drive events that deliver the viral genome to the cytoplasm of a host cell. The extraction of the E1 glycoprotein from virus membranes with detergent results in disulfide-bridge rearrangement and the collapse of the protein to a number of low-energy, non-native configurations. In a new approach to the production of membrane-free membrane glycoproteins, furin protease recognition motifs were installed at various positions in the E1 glycoprotein ectodomain. Proteins containing the furin-sensitive sites undergo normal folding and assembly in the endoplasmic reticulum and only experience the consequence of the mutation during transport to the cell surface. Processing by furin in the Golgi results in the release of the protein from the membrane. Processing of the proteins also impacts the envelopment of the nucleocapsid in the modified plasma membrane. This technique provides a unique method for studying the mechanism of virus assembly and protein structure without altering crucial early events in protein assembly, folding, and maturation. [Copyright &y& Elsevier]

Sindbis virus virions incorporating uncleaved precursor envelope protein PE2 bind efficiently to cell-surface heparan sulfate (HS) because the furin cleavage site (a consensus HS-binding domain) is retained in the mature virus particle. However, they are essentially nonviable. Resuscitating mutations selected in the E3 or E2 protein preserve the PE2 noncleaving phenotype and HS binding, but facilitate fusion, and thereby restore wild-type infectivity on cultured cells. Here, we have demonstrated that the resuscitated PE2 noncleaving virus was almost avirulent in vivo, but mutated during the infection. Mutants had increased virulence and cleavage of PE2, with reduced HS binding capacity. We hypothesize that HS binding leads to sequestration of PE2 noncleaving virus particles and suppression of serum viremia, thereby selecting for evolution of the virus into a PE2-cleaving, low HS-binding phenotype. [Copyright &y& Elsevier]

Alphavirus transducing systems (ATSs) are alphavirus-based tools for expressing genes in insects. Here we describe an ATS (5′dsMRE16ic) based entirely on Sindbis MRE16 virus. GFP expression was used to characterize alimentary tract infections and dissemination in three Culicine and two Lepidopteran species. Following per os infection, 5′dsMRE16ic-EGFP efficiently infected Aedes aegypti and Culex tritaeniorhynchus, but not Culex pipiens pipiens. Ae. aegypti clearly showed accumulation of green fluorescent protein (GFP) in the posterior midgut and foregut/midgut junction within 2–3 days postinfection. Following parenteral infection of larvae, Bombyx mori had extensive GFP expression in larvae and adults, but Manduca sexta larvae were mostly resistant. 5′dsMRE16ic should be a valuable tool for gene expression in several important insect species that are otherwise difficult to manipulate genetically. [ABSTRACT FROM AUTHOR]

Poly(ADP-ribose) polymerase-1 (PARP-1) is a chromatin-associated enzyme that is activated by DNA strand breaks and catalyzes the transfer of ADP-ribose groups from NAD to itself and other nuclear proteins. Although caspase-mediated PARP-1 cleavage occurs during almost all forms of apoptosis, the contribution of PARP-1 activation and cleavage to this cell death process remains unclear. Using immortalized fibroblasts from wild-type (PARP-1+/+) and PARP-1 knockout (PARP-1−/−) mice, and a mouse neuroblastoma cell line (N18), the role that poly(ADP-ribosyl)ation plays in Sindbis virus (SV)-induced apoptosis was examined. Robust PARP-1 activation occurred in SV-infected cells prior to morphologic changes associated with apoptotic cell death and PARP-1 activity ceased simultaneously with caspase-3 activation and PARP-1 proteolysis. PARP-1 activity was maximal before detectable DNA fragmentation, but was absent when DNA damage was most intense. SV and staurosporine-induced cell death was delayed in fibroblasts lacking PARP-1 activity, suggesting that PARP-1 activation contributes to apoptotic cell death induced by these stimuli. SV replication was not affected by lack of PARP-1 activity, but DNA fragmentation and caspase-3 activation were delayed and occurred at lower levels in PARP-1-deficient fibroblasts. Early virus-induced PARP-1 activation may represent a novel way by which cells signal to the nucleus to regulate protein function by poly(ADP-ribosyl)ation in response to virus infection. [Copyright &y& Elsevier]

Abstract Aedes aegypti were injected intrathoracically with double subgenomic Sindbis (dsSIN) viruses with inserted sequences derived from the genome of one or more of the four dengue (DEN) virus serotypes. Mosquitoes were highly resistant to challenge with homologous DEN viruses from which the effector sequences were derived, and resistance to DEN viruses was independent of the orientation of the effector RNA. dsSIN viruses designed to express RNA derived from the premembrane coding region of DEN-2 prevented the accumulation of DEN2 RNA, and C6/36 cells were highly resistant to DEN-2 virus when challenged at 2, 5 or 8 days after the initial dsSIN virus infections, even though the dsSIN-derived RNA had sharply declined at the later time points. Initiation of resistance occurred prior to or within the first 8 h after challenge with DEN-2 virus. We conclude that DEN viruses are inhibited by a mechanism similar to post-transcriptional gene silencing (PTGS) or RNA interference (RNAi) phenomena described in plants and invertebrates, respectively. The potential occurrence of PTGS or RNAi in mosquitoes and mosquito cells suggests new ways of inhibiting the replication of arthropod-borne viruses in mosquito vectors, studying vector–virus interactions, and silencing endogenous mosquito genes. [ABSTRACT FROM AUTHOR]

Bcl-2 oncogene expression plays a role in the establishment of persistent viral infection by blocking virus-induced apoptosis. This might be achieved by preventing virus-induced activation of caspase-3, an IL-1β-converting enzyme (ICE)-like cysteine protease that has been implicated in the death effector phase of apoptosis. Contrary to this model, we show that three cell types highly overexpressing functional Bcl-2 displayed caspase-3 activation and underwent apoptosis in response to infection with alphaviruses Semliki Forest and Sindbis as efficiently as vector control counterparts. In all three cell types, overexpressed 26 kDa Bcl-2 was cleaved into a 23 kDa protein. Antibody epitope mapping revealed that cleavage occurred at one or two target sites for caspases within the amino acid region YEWD31· AGD34· A, removing the N-terminal BH4 region known to be essential for the death-protective activity of Bcl-2. Preincubation of cells with the caspase inhibitor Z-VAD prevented Bcl-2 cleavage and partially restored the protective activity of Bcl-2 against virus-induced apoptosis. Moreover, a murine Bcl-2 mutant having Asp31, Asp34 and Asp36 substituted by Glu was resistant to proteolytic cleavage and abrogated apoptosis following virus infection. These findings indicate that alphaviruses can trigger a caspase­mediated inactivation of Bcl-2 in order to evade the death protection imposed by this survival factor. [ABSTRACT FROM AUTHOR]

RNA of Kyzylagach virus (KYZV), a Sindbis-like mosquito-borne alphavirus from Western equine encephalitis virus complex, was detected in four pools (out of 221 pools examined), encompassing 10,784 female Culex modestus mosquitoes collected at a fishpond in south Moravia, Czech Republic, with a minimum infection rate of 0.04%. This alphavirus was never detected in Central Europe before. [ABSTRACT FROM AUTHOR]

Summary The compendium of RNA-binding proteins (RBPs) has been greatly expanded by the development of RNA-interactome capture (RIC). However, it remained unknown if the complement of RBPs changes in response to environmental perturbations and whether these rearrangements are important. To answer these questions, we developed "comparative RIC" and applied it to cells challenged with an RNA virus called sindbis (SINV). Over 200 RBPs display differential interaction with RNA upon SINV infection. These alterations are mainly driven by the loss of cellular mRNAs and the emergence of viral RNA. RBPs stimulated by the infection redistribute to viral replication factories and regulate the capacity of the virus to infect. For example, ablation of XRN1 causes cells to be refractory to SINV, while GEMIN5 moonlights as a regulator of SINV gene expression. In summary, RNA availability controls RBP localization and function in SINV-infected cells. Graphical Abstract Highlights • A quarter of the RBPome changes upon SINV infection • Alterations in RBP activity are largely explained by changes in RNA availability • Altered RBPs are crucial for viral infection efficacy • GEMIN5 binds to the 5′ end of SINV RNAs and regulates viral gene expression Garcia-Moreno, Noerenberg, Ni, and colleagues developed "comparative RNA-interactome capture" to analyze the RNA-bound proteome during virus infection. More than 200 cellular RNA-binding proteins change their binding activity in response to this challenge, mainly driven by transcript availability. Many of these RNA-binding proteins regulate viral replication and can be targeted to influence infection outcome. [ABSTRACT FROM AUTHOR]