Search

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]

Relevance. Natural and climatic conditions, a variety of species composition of arthropods and vertebrates determine the possibility of circulation of arboviruses in the Volgograd region. The existence of natural foci of some arbovirus infections and the possibility of the formation of others suggests the need for annual monitoring of the causative agents of arbovirus diseases. Аim. Evaluation of the results of monitoring of arbovirus infections in the Volgograd region in 2019.Materials and methods: 806 blood serum samples from donors, 44 blood serum samples from febrile sick people, 300 blood serum samples from horses and 94 pools of blood-sucking mosquitoes were examined by immunofernal analysis. Result of the study of serum samples from donors in the Volgograd region, in 140 (17.4%) of 806 were found to have antibodies to the pathogen of West Nile fever (in 35 (4.3%) – IgM, in 105 (13.0%) – IgG), in 7 (2.2%) of 319 – to the Crimean hemorrhagic fever virus (in 4 (1.3%) – IgM, in 3 (0.9%) –IgG), and in 7 (2.9%) of 240 – IgG to the viruses of the California serogroup. Specific antibodies against viruses of Sindbis, Batai and Uukuniemi in the samples was not detected. The largest number of positive samples with the presence of IgG and IgM to the West Nile virus was found among residents of Volgograd (61 out of 240, 25.4%) and Volzhsky (25 out of 100, 25, 0%). Among 44 blood serums of febrile patients, 1 sample (2.3%) was found to contain an antigen of the Sindbis virus, and 2 samples (4.5%) – antigens California serogroup viruses. Specific immunoglobulins against West Nile virus were detected in 84 (28%) of 300 blood serums of farm animals (horses). In the study of 94 samples of field material (blood-sucking mosquitoes), West Nile virus antigen was detected in 14 (14.9%), Sindbis virus – in one sample (1.0%), Batai virus – in four samples (4.2%). Conclusions: the obtained results, along with the circulation of West Nile virus and Crimean hemorrhagic fever virus virus in the Volgograd region, indicate the presence of Sindbis, Batai and California serogroup viruses and necessitate further study of their role in the infectious pathology of the population.

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.

Despite remarkable responses to cancer immunotherapy in a subset of patients, many patients remain resistant to therapies. It is now clear that elevated levels of tumor-infiltrating T cells as well as a systemic anti-tumor immune response are requirements for successful immunotherapies. However, the tumor microenvironment imposes an additional resistance mechanism to immunotherapy. We have developed a practical and improved strategy for cancer immunotherapy using an oncolytic virus and anti-OX40. This strategy takes advantage of a preexisting T cell immune repertoire in vivo, removing the need to know about present tumor antigens. We have shown in this study that the replication-deficient oncolytic Sindbis virus vector expressing interleukin-12 (IL-12) (SV.IL12) activates immune-mediated tumor killing by inducing OX40 expression on CD4 T cells, allowing the full potential of the agonistic anti-OX40 antibody. The combination of SV.IL12 with anti-OX40 markedly changes the transcriptome signature and metabolic program of T cells, driving the development of highly activated terminally differentiated effector T cells. These metabolically reprogrammed T cells demonstrate enhanced tumor infiltration capacity as well as anti-tumor activity capable of overcoming the repressive tumor microenvironment. Our findings identify SV.IL12 in combination with anti-OX40 to be a novel and potent therapeutic strategy that can cure multiple types of low-immunogenic solid tumors.

Volgograd region is considered to be endemic West Nile fever (WNF) area due to the established circulation of the West Nile virus (WNV) therein. Some previous independent studies examining samples collected on the territory of the Volgograd region revealed markers related to the California serogroup (CSG), Sindbis and Ukuniemi viruses. WNF, CSG, Sindbis and Ukuniemi fever mainly being asymptomatic posing thereby a threat of virus spread due to transfusiological manipulations along with vector-borne transmission are manifested by diverse clinical signs. The study was aimed at detection of antibodies specific to West Nile, tick-borne encephalitis, California serogroup (Tyaginya, Inko), Sidbis, and Ukuniemi viruses in blood donors from the Volgograd region. For this, 404 blood sera samples collected from blood donors residing in the Volgograd as well as the Volgograd region were examined by ELISA. It was found that percentage of blood serum samples positive for arbovirus-specific antibodies was 18.32%. Among the, 67 out of 404 (16.58%) samples contained anti-WNV antibodies, 3 out of 279 (1.08%) samples — to tick-borne encephalitis virus, 1 out of 92 (1.09%) — to California serogroup fever and Ukuniemi viruses, and 2 out of 92 (2.17%) — to Sindbis fever virus. Importantly, the peak number of IgG and IgM WNV-positive samples was found among residents of Volgograd (29 out of 110, 26%) and Oktyabrsky district (7 out of 25, 28%). In addition, anti-Sindbis, Ukuniemi and CSG virus antibodies were detected in blood serum samples from blood donors residing in the Kalachevsky region and the city of Volgograd. Analyzing age-related distribution and percentage of seropositive subjects in each age group showed as follows: the lowest percentage (14.5%) was positive for anti-WNV, Sindbis, Ukuniemi and CSG virus antibodies in blood donors aged 32–41 years, whereas the peak percentage (25%) — in the subjects aged 52–61 years. Thus, virus specific antibodies detected in healthy individuals in the aforementioned region evidence about potential recovery after asymptomatic infections. In this regard, further research is required to determine a role of the such arboviruses in the structure of regional infectious diseases. The data obtained suggest a need to continue serological arbovirus monitoring in the Volgograd region.

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.

The article presents the discussion on globalization and climate change where mosquito borne pathogens posing a significant health threat to humans. Topics include sudden onset of headache and arthralgia or arthritis showing rash on the thorax and extremities; and disease remaining unrecognized in humans and misdiagnosed as a fever of unknown etiology.

Purpose of Review: Persistent joint pain is a common manifestation of arthropod-borne viral infections and can cause long-term disability. We review the epidemiology, pathophysiology, diagnosis, and management of arthritogenic alphavirus infection. Recent findings: The global re-emergence of alphaviral outbreaks has led to an increase in virus-induced arthralgia and arthritis. Alphaviruses, including Chikungunya, O'nyong'nyong, Sindbis, Barmah Forest, Ross River, and Mayaro viruses, are associated with acute and/or chronic rheumatic symptoms. Identification of Mxra8 as a viral entry receptor in the alphaviral replication pathway creates opportunities for treatment and prevention. Recent evidence suggesting virus does not persist in synovial fluid during chronic chikungunya infection indicates that immunomodulators may be given safely. Summary: The etiology of persistent joint pain after alphavirus infection is still poorly understood. New diagnostic tools along and evidence-based treatment could significantly improve morbidity and long-term disability. [ABSTRACT FROM AUTHOR]

ABSTRACT DEAD box RNA helicases regulate diverse facets of RNA biology. Proteins of this family carry out essential cellular functions, and emerging literature is revealing additional roles in immune defense. Using RNA interference screening, we identified an evolutionarily conserved antiviral role for the helicase DDX56 against the alphavirus Sindbis virus (SINV), a mosquito-transmitted pathogen that infects humans. Depletion of DDX56 enhanced infection in Drosophila and human cells. Furthermore, we found that DDX56 also controls the emerging alphavirus chikungunya virus (CHIKV) through an interferon-independent mechanism. Using cross-linking immunoprecipitation (CLIP-Seq), we identified a predicted stem-loop on the viral genomic RNA bound by DDX56. Mechanistically, we found that DDX56 levels increase in the cytoplasm during CHIKV infection. In the cytoplasm, DDX56 impacts the earliest step in the viral replication cycle by binding and destabilizing the incoming viral genomic RNA, thereby attenuating infection. Thus, DDX56 is a conserved antiviral RNA binding protein that controls alphavirus infection. IMPORTANCE Arthropod-borne viruses are diverse pathogens and include the emerging virus chikungunya virus, which is associated with human disease. Through genetic screening, we found that the conserved RNA binding protein DDX56 is antiviral against chikungunya virus in insects and humans. DDX56 relocalizes from the nucleus to the cytoplasm, where it binds to a stem-loop in the viral genome and destabilizes incoming genomes. Thus, DDX56 is an evolutionarily conserved antiviral factor that controls alphavirus infection.

ABSTRACT Despite their exceptional potencies, the broad tropism of most commonly used lentivirus (LV) vectors limits their use for targeted gene delivery in vivo. We hypothesized that we could improve the specificity of LV targeting by coupling (i) reduction of their binding to off-target cells with (ii) redirection of the vectors with a bispecific antibody (bsAb) that binds both LV and receptors on target cells. As a proof of concept, we pseudotyped nonreplicating LV using a mutated Sindbis envelope (mSindbis) with ablated binding to native receptors, while retaining the capacity to facilitate efficient fusion and endosomal escape. We then evaluated the transduction potencies of the mSindbis LV for HER2-positive (HER2+) (SKBR3) breast and HER2-negative (HER2−) (A2780) cells when redirected with different bsAbs. mSindbis LV alone failed to induce appreciable green fluorescent protein (GFP) expression in either cell. When mixed with HER2-targeting bsAb, mSindbis LV was exceptionally potent, transducing 12% to 16% of the SKBR3 cells at a multiplicity of infection (MOI [ratio of viral genome copies to target cells]) of 3. Transduction was highly specific, resulting in ∼50-fold-greater selectivity toward SKBR3 cells versus A2780 cells. Redirecting mSindbis LV led to a 10-fold improvement in cell-specific targeting compared to redirecting wild-type Sindbis LV with the same bsAb, underscoring the importance of ablating native virus tropism in order to maximize targeting specificity. The redirection of mutated LV using bsAb represents a potent and highly versatile platform for targeted gene therapy. IMPORTANCE The goal of gene therapy is specific delivery and expression of therapeutic genes to target cells and tissues. Common lentivirus (LV) vectors are efficient gene delivery vehicles but offer little specificity. Here, we report an effective and versatile strategy to redirect LV to target cells using bispecific antibodies (bsAbs) that bind both cell receptors and LV envelope domains. Importantly, we ablated the native receptor binding of LV to minimize off-target transduction. Coupling bsAb specificity and ablated native LV tropism synergistically enhanced the selectivity of our targeted gene delivery system. The modular nature of our bsAb-based redirection enables facile targeting of the same LV to diverse tissues/cells. By abrogating the native broad tropism of LV, our bsAb-LV redirection strategy may enable lentivirus-based gene delivery in vivo, expanding the current use of LV beyond ex vivo applications.

Arboviruses including alphavirus are responsible for most emerging infectious diseases worldwide. Recent outbreaks of chikungunya virus serve as a stark reminder to their pathogenic potential. There are no vaccines or therapeutics currently available to contain alphavirus outbreaks. In this study we evaluated the effect of immunomodulatory CpG ODN on the clinical progression of neurotropic Sindbis virus infection. Neonatal C57Bl-6 mice challenged with Sindbis virus AR339 (25 PFU Subcutaneous) infect neurons in the CNS leading to the development of ataxia, seizures, paralysis, and death. We show that systemic administration of CpG ODN modulates the cytokine and chemokine gene expression levels in the CNS and ultimately protects neonatal mice from lethal neurotropic infection. The protection conferred by CpG ODN is controlled by innate immune response and T and B cells were dispensable. Further, protection required Type I, Type II interferons, and TNF as well as functional NK cells, but did not involve iNOS. This study confirms that administration of innate immune modulators can be used as a strategy to boost host innate immune responses and protect against neurotropic viruses reducing their pathogenic footprint.

Arboviruses including alphavirus are responsible for most emerging infectious diseases worldwide. Recent outbreaks of chikungunya virus serve as a stark reminder to their pathogenic potential. There are no vaccines or therapeutics currently available to contain alphavirus outbreaks. In this study we evaluated the effect of immunomodulatory CpG ODN on the clinical progression of neurotropic Sindbis virus infection. Neonatal C57Bl-6 mice challenged with Sindbis virus AR339 (25 PFU Subcutaneous) infect neurons in the CNS leading to the development of ataxia, seizures, paralysis, and death. We show that systemic administration of CpG ODN modulates the cytokine and chemokine gene expression levels in the CNS and ultimately protects neonatal mice from lethal neurotropic infection. The protection conferred by CpG ODN is controlled by innate immune response and T and B cells were dispensable. Further, protection required Type I, Type II interferons, and TNF as well as functional NK cells, but did not involve iNOS. This study confirms that administration of innate immune modulators can be used as a strategy to boost host innate immune responses and protect against neurotropic viruses reducing their pathogenic footprint. [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]

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.

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]

Arthropod-borne viruses (arboviruses) may cause severe emerging and re-emerging infectious diseases, which pose a significant threat to human and animal health in the world today. These infectious diseases range from mild febrile illnesses, arthritis, and encephalitis to haemorrhagic fevers. It is postulated that certain environmental factors, vector competence, and host susceptibility have a major impact on the ecology of arboviral diseases. Presently, there is a great interest in the emergence of Alphaviruses because these viruses, including Chikungunya virus, O'nyong'nyong virus, Sindbis virus, Ross River virus, and Mayaro virus, have caused outbreaks in Africa, Asia, Australia, Europe, and America. Some of these viruses are more common in the tropics, whereas others are also found in temperate regions, but the actual factors driving Alphavirus emergence and re-emergence remain unresolved. Furthermore, little is known about the transmission dynamics, pathophysiology, genetic diversity, and evolution of circulating viral strains. In addition, the clinical presentation of Alphaviruses may be similar to other diseases such as dengue, malaria, and typhoid, hence leading to misdiagnosis. However, the typical presence of arthritis may distinguish between Alphaviruses and other differential diagnoses. The absence of validated diagnostic kits for Alphaviruses makes even routine surveillance less feasible. For that purpose, this review describes the occurrence, genetic diversity, clinical characteristics, and the mechanisms involving Alphaviruses causing arthritis in humans. This information may serve as a basis for better awareness and detection of Alphavirus-caused diseases during outbreaks and in establishing appropriate prevention and control measures.

We report a highly efficient, simple and non-infective method for labeling individual long-range projection neurons in a specific location with enough sparseness and intensity to allow complete and unambiguous reconstructions of their entire axonal tree. The method is based on the in vivo transfection of a large RNA construct that drives the massive expression of green fluorescent protein. The method combines two components: injection of a small volume of a hyperosmolar NaCl solution containing the Pal-eGFP-Sindbis RNA construct (Furuta et al., 2001), followed by the application of high-frequency electric current pulses through the micropipette tip. We show that, although each component alone increases transfection efficacy, compared to simple volume injections of standard RNA solution, the highest efficacy (85.7%) is achieved by the combination of both components. In contrast with the infective viral Sindbis vector, RNA transfection occurs exclusively at the position of the injection micropipette tip. This method simplifies consistently labeling one or a few isolated neurons per brain, a strategy that allows unambiguously resolving and quantifying the brain-wide and often multi-branched monosynaptic circuits created by long-range projection neurons.

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]

We report a highly efficient, simple, and non-infective method for labeling individual long-range projection neurons (LRPNs) in a specific location with enough sparseness and intensity to allow complete and unambiguous reconstructions of their entire axonal tree. The method is based on the "in vivo" transfection of a large RNA construct that drives the massive expression of green fluorescent protein. The method combines two components: injection of a small volume of a hyperosmolar NaCl solution containing the Pal-eGFP-Sindbis RNA construct (Furuta et al., 2001), followed by the application of high-frequency electric current pulses through the micropipette tip. We show that, although each component alone increases transfection efficacy, compared to simple volume injections of standard RNA solution, the highest efficacy (85.7%) is achieved by the combination of both components. In contrast with the infective viral Sindbis vector, RNA transfection occurs exclusively at the position of the injection micropipette tip. This method simplifies consistently labeling one or a few isolated neurons per brain, a strategy that allows unambiguously resolving and quantifying the brain-wide and often multi-branched monosynaptic circuits created by LRPNs. [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]

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.

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.

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]

DEAD box RNA helicases regulate diverse facets of RNA biology. Proteins of this family carry out essential cellular functions, and emerging literature is revealing additional roles in immune defense. Using RNA interference screening, we identified an evolutionarily conserved antiviral role for the helicase DDX56 against the alphavirus Sindbis virus (SINV), a mosquito-transmitted pathogen that infects humans. Depletion of DDX56 enhanced infection in Drosophila and human cells. Furthermore, we found that DDX56 also controls the emerging alphavirus chikungunya virus (CHIKV) through an interferon-independent mechanism. Using cross-linking immunoprecipitation (CLIP-Seq), we identified a predicted stem-loop on the viral genomic RNA bound by DDX56. Mechanistically, we found that DDX56 levels increase in the cytoplasm during CHIKV infection. In the cytoplasm, DDX56 impacts the earliest step in the viral replication cycle by binding and destabilizing the incoming viral genomic RNA, thereby attenuating infection. Thus, DDX56 is a conserved antiviral RNA binding protein that controls alphavirus infection. IMPORTANCE Arthropod-borne viruses are diverse pathogens and include the emerging virus chikungunya virus, which is associated with human disease. Through genetic screening, we found that the conserved RNA binding protein DDX56 is antiviral against chikungunya virus in insects and humans. DDX56 relocalizes from the nucleus to the cytoplasm, where it binds to a stem-loop in the viral genome and destabilizes incoming genomes. Thus, DDX56 is an evolutionarily conserved antiviral factor that controls alphavirus infection., (Copyright © 2020 Taschuk et al.)

Despite remarkable responses to cancer immunotherapy in a subset of patients, many patients remain resistant to therapies. It is now clear that elevated levels of tumor-infiltrating T cells as well as a systemic anti-tumor immune response are requirements for successful immunotherapies. However, the tumor microenvironment imposes an additional resistance mechanism to immunotherapy. We have developed a practical and improved strategy for cancer immunotherapy using an oncolytic virus and anti-OX40. This strategy takes advantage of a preexisting T cell immune repertoire in vivo , removing the need to know about present tumor antigens. We have shown in this study that the replication-deficient oncolytic Sindbis virus vector expressing interleukin-12 (IL-12) (SV.IL12) activates immune-mediated tumor killing by inducing OX40 expression on CD4 T cells, allowing the full potential of the agonistic anti-OX40 antibody. The combination of SV.IL12 with anti-OX40 markedly changes the transcriptome signature and metabolic program of T cells, driving the development of highly activated terminally differentiated effector T cells. These metabolically reprogrammed T cells demonstrate enhanced tumor infiltration capacity as well as anti-tumor activity capable of overcoming the repressive tumor microenvironment. Our findings identify SV.IL12 in combination with anti-OX40 to be a novel and potent therapeutic strategy that can cure multiple types of low-immunogenic solid tumors., (© 2020 The Author(s).)

Despite their exceptional potencies, the broad tropism of most commonly used lentivirus (LV) vectors limits their use for targeted gene delivery in vivo We hypothesized that we could improve the specificity of LV targeting by coupling (i) reduction of their binding to off-target cells with (ii) redirection of the vectors with a bispecific antibody (bsAb) that binds both LV and receptors on target cells. As a proof of concept, we pseudotyped nonreplicating LV using a mutated Sindbis envelope (mSindbis) with ablated binding to native receptors, while retaining the capacity to facilitate efficient fusion and endosomal escape. We then evaluated the transduction potencies of the mSindbis LV for HER2-positive (HER2 + ) (SKBR3) breast and HER2-negative (HER2 - ) (A2780) cells when redirected with different bsAbs. mSindbis LV alone failed to induce appreciable green fluorescent protein (GFP) expression in either cell. When mixed with HER2-targeting bsAb, mSindbis LV was exceptionally potent, transducing 12% to 16% of the SKBR3 cells at a multiplicity of infection (MOI [ratio of viral genome copies to target cells]) of 3. Transduction was highly specific, resulting in ∼50-fold-greater selectivity toward SKBR3 cells versus A2780 cells. Redirecting mSindbis LV led to a 10-fold improvement in cell-specific targeting compared to redirecting wild-type Sindbis LV with the same bsAb, underscoring the importance of ablating native virus tropism in order to maximize targeting specificity. The redirection of mutated LV using bsAb represents a potent and highly versatile platform for targeted gene therapy. IMPORTANCE The goal of gene therapy is specific delivery and expression of therapeutic genes to target cells and tissues. Common lentivirus (LV) vectors are efficient gene delivery vehicles but offer little specificity. Here, we report an effective and versatile strategy to redirect LV to target cells using bispecific antibodies (bsAbs) that bind both cell receptors and LV envelope domains. Importantly, we ablated the native receptor binding of LV to minimize off-target transduction. Coupling bsAb specificity and ablated native LV tropism synergistically enhanced the selectivity of our targeted gene delivery system. The modular nature of our bsAb-based redirection enables facile targeting of the same LV to diverse tissues/cells. By abrogating the native broad tropism of LV, our bsAb-LV redirection strategy may enable lentivirus-based gene delivery in vivo , expanding the current use of LV beyond ex vivo applications., (Copyright © 2020 Parker et al.)

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]

Arthropod-borne viruses (arboviruses) may cause severe emerging and re-emerging infectious diseases, which pose a significant threat to human and animal health in the world today. These infectious diseases range from mild febrile illnesses, arthritis, and encephalitis to haemorrhagic fevers. It is postulated that certain environmental factors, vector competence, and host susceptibility have a major impact on the ecology of arboviral diseases. Presently, there is a great interest in the emergence of Alphaviruses because these viruses, including Chikungunya virus, O'nyong'nyong virus, Sindbis virus, Ross River virus, and Mayaro virus, have caused outbreaks in Africa, Asia, Australia, Europe, and America. Some of these viruses are more common in the tropics, whereas others are also found in temperate regions, but the actual factors driving Alphavirus emergence and re-emergence remain unresolved. Furthermore, little is known about the transmission dynamics, pathophysiology, genetic diversity, and evolution of circulating viral strains. In addition, the clinical presentation of Alphaviruses may be similar to other diseases such as dengue, malaria, and typhoid, hence leading to misdiagnosis. However, the typical presence of arthritis may distinguish between Alphaviruses and other differential diagnoses. The absence of validated diagnostic kits for Alphaviruses makes even routine surveillance less feasible. For that purpose, this review describes the occurrence, genetic diversity, clinical characteristics, and the mechanisms involving Alphaviruses causing arthritis in humans. This information may serve as a basis for better awareness and detection of Alphavirus-caused diseases during outbreaks and in establishing appropriate prevention and control measures.

RNA replicons are an emerging platform for engineering synthetic biological systems. Replicons self-amplify, can provide persistent high-level expression of proteins even from a small initial dose, and, unlike DNA vectors, pose minimal risk of chromosomal integration. However, no quantitative model sufficient for engineering levels of protein expression from such replicon systems currently exists. Here, we aim to enable the engineering of multigene expression from more than one species of replicon by creating a computational model based on our experimental observations of the expression dynamics in single- and multireplicon systems. To this end, we studied fluorescent protein expression in baby hamster kidney (BHK-21) cells using a replicon derived from Sindbis virus (SINV). We characterized expression dynamics for this platform based on the dose-response of a single species of replicon over 50 h and on a titration of two cotransfected replicons expressing different fluorescent proteins. From this data, we derive a quantitative model of multireplicon expression and validate it by designing a variety of three-replicon systems, with profiles that match desired expression levels. We achieved a mean error of 1.7-fold on a 1000-fold range, thus demonstrating how our model can be applied to precisely control expression levels of each Sindbis replicon species in a system.

Viruses have evolved to exploit the vast complexity of cellular processes for their success within the host cell. The entry mechanisms of enveloped viruses (viruses with a surrounding outer lipid bilayer membrane) are usually classified as being either endocytotic or fusogenic. Different mechanisms have been proposed for Alphavirus entry and genome delivery. Indirect observations led to a general belief that enveloped viruses can infect cells either by protein-assisted fusion with the plasma membrane in a pH-independent manner or by endocytosis and fusion with the endocytic vacuole in a low-pH environment. The mechanism of Alphavirus penetration has been recently revisited using direct observation of the processes by electron microscopy under conditions of different temperatures and time progression. Under conditions nonpermissive for endocytosis or any vesicular transport, events occur which allow the entry of the virus genome into the cells. When drug inhibitors of cellular functions are used to prevent entry, only ionophores are found to significantly inhibit RNA delivery. Arboviruses are agents of significant human and animal disease; therefore, strategies to control infections are needed and include development of compounds which will block critical steps in the early infection events. It appears that current evidence points to an entry mechanism, in which alphaviruses infect cells by direct penetration of cell plasma membranes through a pore structure formed by virus and, possibly, host proteins., (© 2015 Elsevier Inc. All rights reserved.)