Search Results (79)

Arthropod-borne viruses (arboviruses) cause a wide range of acute and chronic diseases and represent a growing global health burden. Although some vaccines exist, antiviral therapies remain limited. Identifying host restriction factors may enable new therapeutic strategies. We previously showed that bacterial effector proteins can serve as tools to uncover innate immune defenses. Here, we used a bacterial effector screen in bat cells to identify host factors restricting the arboviruses Sindbis virus (SINV) and O’nyong’nyong virus (ONNV). Several effectors enhanced infection by both viruses. However, we found the Shigella flexneri-encoded E3 ubiquitin ligase IpaH2 to selectively promote SINV replication. IpaH2 enhanced SINV infection across multiple mammalian cell lines, suggesting that it targets a conserved antiviral mechanism, and this effect required IpaH2 E3 ubiquitin ligase activity. Screening of putative IpaH2 host targets identified via ubiquitin-activated interaction trap (UBAIT) assays revealed the host factors ATP-binding cassette sub-family F member 3 (ABCF3) and Plakophilin-2 (PKP2) to play roles in restricting SINV infection. While ABCF3 broadly restricted multiple viruses, PKP2 specifically limited SINV replication, indicating a virus-specific restriction factor. These findings demonstrate that bacterial effector screening can identify both broadly acting and virus-specific host defenses, providing insight into antiviral mechanisms and potential therapeutic targets. Full article

Alphaviruses are mosquito-borne viruses that can infect the central nervous system (CNS) and cause encephalomyelitis, which is a rare but dangerous complication from infection. In mice, this can be studied in a model of infection with Sindbis virus (SINV), which infects neurons and causes neurological disease. Due to the non-renewable nature of neurons, the immune response in the CNS is specialized to prevent neuronal damage or death, even if they are infected. Therefore, insights into the nuances of antiviral immunity in the CNS provide a better understanding of disease pathogenesis and mechanisms of recovery. Type I interferons (IFNs) are critically important for survival; they are an innate antiviral defense mechanism that consists mainly of IFNα and IFNβ. Although both use the same receptor, type-specific differences between IFNα and IFNβ have been described in other contexts. To this end, Ifnb^−/− mice were used to elucidate the role of IFNβ in recovery from alphavirus encephalomyelitis. IFNβ-deficient mice have intact IFNα expression and downstream signaling, but symptomatic disease occurs earlier and is more severe. This is accompanied by increased virus replication in the early stages of infection. Microgliosis is reduced in Ifnb^−/− mice compared to wildtype, but inflammatory cytokine/chemokine levels are higher and associated with alterations in monocyte and NK cell recruitment into the CNS. Ifnb^−/− mice have no deficiencies in the expression of factors known to be required for viral clearance. Therefore, IFNβ modulates the early stages of the immune response and facilitates restriction of virus replication, contributing to delayed disease onset. Full article

The re-emergence of alphaviruses (family Togaviridae), such as chikungunya virus, poses significant public health risks, with direct impact on quality of life and work productivity. There are no approved antiviral drugs for the treatment of infections with alphaviruses. Several alphaviruses are classified as risk group 3 agents which require handling in high-containment laboratories. To facilitate antiviral screening efforts against alphaviruses, we established a high-throughput antiviral screening assay using reporter Sindbis virus [SINV-GFP; expresses the green fluorescent protein] as a surrogate model for more pathogenic alphaviruses. The assay has strong reproducibility and was validated by reference small-molecule antivirals with various mechanisms of action. The use of high-content imaging as a readout, as demonstrated here, allows for high-throughput antiviral screening and provides a tool for early-stage antiviral discovery against emerging alphavirus threats at a lower biosafety level. Full article

Sindbis virus (SINV) is a mosquito-borne alphavirus capable of causing neurological and immunological symptoms in humans, yet its effects on neural/immune systems remain insufficiently characterized. This study aimed to examine SINV replication, UV-C light inactivation, apoptosis induction, and immune gene modulation in human SH-SY5Y neuroblastoma cells. Following viral adaptation and infectious dose determination, SINV replication and inactivation were assessed using RT-qPCR and dsRNA immunofluorescence. Apoptotic markers (caspase-3, Bax, Bcl-2) were analyzed by immunofluorescence and immune genes expression kinetics (TLR3/7, RIGI, MDA5, IL-1β, IL-6, TNFα, IL-10, IFNβ and β-catenin) were measured at defined time points post-infection by RT-qPCR. SH-SY5Y cells supported productive SINV infection, with viral RNA detectable as early as 3 hpi and marked cytopathic effects by 24 hpi. A custom-built UV-C chamber achieved complete viral inactivation following 3 × 30 s exposures. We observed SINV time-course replication and UV-C inactivation with conspicuous morphological alterations in SH-SY5Y cells. Furthermore, SINV triggered caspase-dependent apoptosis and robust transcriptional upregulation of innate immune genes, peaking between 12–16 hpi and declining by 30 hpi. These findings elucidate the temporal dynamics of SINV replication, cell death mechanisms, and immune activation in a neuronal context, contributing to a better understanding of SINV neuropathogenesis. Full article

Sindbis virus (SINV), a prototype of the Alphavirus genus (family Togaviridae), is a globally distributed arbovirus causing febrile rash and debilitating arthritis in humans. Viral structural proteins—capsid (C), E1, and E2—are fundamental to the virion’s architecture, mediating all stages from assembly to host cell entry and pathogenesis, thus representing critical targets for study. This review consolidates the historical and current understanding of SINV structural biology, tracing progress from early microscopy to recent high-resolution cryo-electron microscopy (cryo-EM) and X-ray crystallography. We detail the virion’s precise T = 4 icosahedral architecture, composed of a nucleocapsid core and an outer glycoprotein shell. Key functional roles tied to protein structure are examined: the capsid’s dual capacity as a serine protease and an RNA-packaging scaffold that interacts with the E2 cytoplasmic tail; the E1 glycoprotein’s function as a class II fusion protein driving membrane fusion; and the E2 glycoprotein’s primary role in receptor binding, which dictates cellular tropism and serves as the main antigenic target. Furthermore, we connect these molecular structures to viral evolution and disease, analyzing how genetic variation among SINV genotypes, particularly in the E2 gene, influences host adaptation, immune evasion, and the clinical expression of arthritogenic and neurovirulent disease. In conclusion, the wealth of structural data on SINV offers a powerful paradigm for understanding alphavirus biology. However, critical gaps persist, including the high-resolution visualization of dynamic conformational states during viral entry and the specific molecular determinants of chronic disease. Addressing these challenges through integrative structural and functional studies is paramount. Such knowledge will be indispensable for the rational design of next-generation antiviral therapies and broadly protective vaccines against the ongoing threat posed by SINV and related pathogenic alphaviruses. Full article

Black flies (Diptera: Simuliidae) are important vectors of pathogens, including filarial nematodes, protozoans, and arboviruses, which significantly impact human and animal health. Although their role in arbovirus transmission has not been as thoroughly studied as that of mosquitoes and ticks, advances in molecular tools, particularly metagenomics, have enabled the identification of non-cultivable viruses, significantly enhancing our understanding of black-fly-borne viral diversity and their public and veterinary health implications. However, these methods can also detect insect-specific viruses (i.e., viruses that are unable to replicate in vertebrate hosts), which may lead to the incorrect classification of black flies as potential vectors. This underscores the need for further research into their ecological and epidemiological roles. This systematic review, conducted following the PRISMA protocol, compiled and analyzed evidence on arbovirus detection in Simuliidae from scientific databases. Several arboviruses were identified in these insects, including vesicular stomatitis virus New Jersey serotype (VSVNJ), Venezuelan equine encephalitis virus (VEEV), and Rift Valley fever virus. Additionally, in vitro studies evaluating the vector competence of Simuliidae for arboviruses such as dengue virus, Murray Valley encephalitis virus, and Sindbis virus were reviewed. These findings provide critical insights into the potential role of black flies in arbovirus transmission cycles, emphasizing their importance as vectors in both public and veterinary health contexts. Full article

Background/Objectives: Our laboratory has been developing a Sindbis viral (SV) vector platform for treatments of several types of cancers. In this study, we assess treatment efficacy for metastatic and immunosuppressive pancreatic cancer. Methods: Orthotopic mouse models were generated by injection of tumor cells into the pancreatic parenchyma. Sindbis vectors were inoculated intraperitoneally. Imaging of tumors was performed by either MRI or in vivo imaging using luciferase. Flow cytometry, multi-immunofluorescence and elispot analysis were performed for certain tumors. Results: SV can infect and reduce pancreatic tumors in three mouse model systems: a model bearing human pancreatic tumors, a highly metastatic model, and a model that reflects the highly immunosuppressive, desmoplastic microenvironment common to human pancreatic cancer. Conclusions: Combination of SV vector expressing IL12 with an immune co-stimulatory agent, anti-OX40, can reduce tumors, facilitate an influx of immune response cells into the tumor microenvironment, and prevent tumors in mice rechallenged with tumor cells promising an effective treatment for pancreatic cancer. Full article

Background/Objectives: Oncolytic viruses infect and kill tumor cells while leaving normal cells unharmed. They are often attenuated through the reduction in their ability to antagonize antiviral defenses, leading to robust replication in tumor cells, which often possess defects in antiviral pathways, while minimizing replication in normal cells. However, not all tumors have defects in their antiviral defenses, and virus replication in these tumors is minimal, thus limiting therapeutic benefits. Therefore, identifying and modulating host factors that regulate virus replication in oncolytic virus-resistant cancer cells, but not normal cells, could lead to increased replication in these tumors and potentially improved therapeutic outcomes. Methods: To identify host factors that modulate oncolytic virus replication in tumor cells, we conducted an RNA interference screen by using a replication-competent library of Sindbis virus recombinants individually enabled with the capacity to elicit RNA interference in host genes via the expression of artificial microRNAs. Since the expression of artificial microRNAs is coupled to virus replication, this results in the selective enrichment of viral clones which express an artificial microRNA that promotes virus replication. Results: By using this approach, the serial passage of the Sindbis virus–artificial microRNA library in a tumor cell line followed by the deep sequencing of the selected viral populations led to the identification of several artificial microRNA sequences that were enriched. Furthermore, the identified artificial miRNA sequences increased the replication of several oncolytic viruses both in vitro and in vivo, ultimately leading to an enhanced therapeutic effect. Conclusions: Altogether, our study highlights the utility of this screening platform in identifying artificial microRNAs that enhance oncolytic virus efficacy. Full article

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that, since its re-emergence in 2004, has become recognised as a major public health concern throughout many tropical and sub-tropical regions of the world. Amongst the insights gained from studies on other alphaviruses, several key determinants of virulence have been identified, including one present at the P3 position in the nsP1/nsP2 cleavage domain of the S.A.AR86 Sindbis (SINV) strain. This strain is associated with neurovirulence in adult mice; however, when a threonine-to-isoleucine substitution is engineered at this P3 position, an attenuated phenotype results. A reverse genetics system was developed to evaluate the phenotype that resulted from the substitution of alanine, present at the P3 position in the wild-type CHIKV clone, with valine. The A533V-mutant CHIKV induced milder disease symptoms in the C57BL/6 mouse model than the wild-type virus, in terms of severity of inflammation, length of viraemic period, and histological changes. Furthermore, the induction of type I IFN occurred more rapidly in both CHIKV-infected cell cultures and the mouse model with the mutant CHIKV. Full article

Babanki virus is a subtype of the Sindbis virus, a widespread arthropod-borne alphavirus circulating in Eurasia, Africa, and Oceania. Characterized by rashes and arthritis, clinical infections due to Sindbis were mainly reported in Africa, Australia, Asia, and Europe. However, its sub-type, Babanki virus, was reported in Northern Europe and Africa, where its epidemiology potential remains poorly understood. The diagnosis of alphaviruses is mainly based on serological testing and conventional PCR methods, which have considerable limits. In this study, we developed a real-time qRT-PCR assay for the detection of Babanki virus. The analytical sensitivity and specificity of the newly established assay were evaluated using in vitro standard RNA and related viruses relevant to the African context, respectively. In addition, its diagnostic sensitivity was assessed using a subset of Babanki virus-positive and -negative mosquito pools collected from the field. The new real-time qRT-PCR assay exhibited a 100% specificity, a 95% detection limit of 1 RNA molecule/reaction, and a diagnostic sensitivity of up to 120 pfu/reaction. This newly established assay could be useful not only for the detection of Babanki virus during epidemics but also in future experimental and surveillance studies focusing on their epidemiology and pathogenicity. Full article

Background/Objectives: Since its emergence in 2019, the rapid spread of SARS-CoV-2 led to the global pandemic. Recent large-scale dengue fever outbreaks overlapped with the COVID-19 pandemic, leading to increased cases of co-infection and posing severe public health risks. Accordingly, the development of effective combined SARS-CoV-2 and dengue virus (DENV) vaccines is necessary to control the spread and prevalence of both viruses. Methods: In this study, we designed Sindbis virus (SINV) replicon-based SARS-CoV-2 and DENV chimeric vaccines using two delivery strategies: DNA-launched self-replicating RNA replicon (DREP) and viral replicon particle (VRP) systems. Results: Cellular and animal experiments confirmed that the vaccines effectively produced viral proteins and elicited strong immunogenicity. These vaccines induced robust immune responses and neutralizing activity against live SARS-CoV-2, DENV1, and DENV2 viruses. In addition, passively transferred sera from BALB/c mice immunized with these vaccines into AG129 mice provided significant protection against lethal DENV2 challenge. The transferred sera protected the mice from physical symptoms, reduced viral loads in the kidney, spleen, liver, and intestine, and prevented DENV2-induced vascular leakage in these tissues. Conclusions: Therefore, combined vaccines based on the SINV replicon system are promising candidates for pandemic control. These results lay a foundation for further development of a safe and effective combination vaccine against SARS-CoV-2 and DENV. Full article

Oral infection of mosquitoes by arboviruses often results in a large degree of variation in the amount of infectious virus between individual mosquitoes, even when the mosquitoes are from inbred laboratory strains. This variability in arbovirus load has been shown to affect virus transmissibility. Previously, our group described population genetic and specific infectivity differences between the virus populations found in high and low titer Aedes aegypti mosquitoes that had been orally infected with Sindbis virus (SINV). In this study, we sought to investigate whether there were also differences in transcriptomic response between these high and low titer mosquitoes. Results from the transcriptomic data analysis showed that more genes involved in antiviral activity, endopeptidase activity, and methyltransferase activity were upregulated in low titer mosquitoes than in high titer mosquitoes, relative to blood-fed controls. Meanwhile, genes involved in ion transport, energy metabolism, acetylation, glycosylation, lipid metabolism, and transport tended to be upregulated in high titer mosquitoes more than in low titer mosquitoes, relative to blood-fed mosquitoes. Overall, genes involved in antiviral activities tended to be upregulated in low titer mosquitoes while genes involved in proviral activities were mostly upregulated in high titer mosquitoes. This study has identified a number of candidate mosquito genes that are putatively associated with SINV titer variability after oral infection of Ae. aegypti, and these can now be investigated in order to ascertain their roles in virus replication and their contributions to determining vector competence. Full article

Hepatocellular carcinoma is a refractory tumor with poor prognosis and high mortality. Many oncolytic viruses are currently being investigated for the treatment of hepatocellular carcinoma. Based on previous studies, we constructed a recombinant GM-CSF-carrying Sindbis virus, named SINV-GM-CSF, which contains a mutation (G to S) at amino acid 285 in the nsp1 protein of the viral vector. The potential of this mutated vector for liver cancer therapy was verified at the cellular level and in vivo, respectively, and the changes in the tumor microenvironment after treatment were also described. The results showed that the Sindbis virus could effectively infect hepatocellular carcinoma cell lines and induce cell death. Furthermore, the addition of GM-CSF enhanced the tumor-killing effect of the Sindbis virus and increased the number of immune cells in the intra-tumor microenvironment during the treatment. In particular, SINV-GM-CSF was able to efficiently kill tumors in a mouse tumor model of hepatocellular carcinoma by regulating the elevation of M1-type macrophages (which have a tumor-resistant ability) and the decrease in M2-type macrophages (which have a tumor-promoting capacity). Overall, SINV-GM-CSF is an attractive vector platform with clinical potential for use as a safe and effective oncolytic virus. Full article

This review article provides a comprehensive overview of a novel Sindbis virus vaccine platform as potential immunotherapy for ovarian cancer patients. Ovarian cancer is the most lethal of all gynecological malignancies. The majority of high-grade serous ovarian cancer (HGSOC) patients are diagnosed with advanced disease. Current treatment options are very aggressive and limited, resulting in tumor recurrences and 50–60% patient mortality within 5 years. The unique properties of armed oncolytic Sindbis virus vectors (SV) in vivo have garnered significant interest in recent years to potently target and treat ovarian cancer. We discuss the molecular biology of Sindbis virus, its mechanisms of action against ovarian cancer cells, preclinical in vivo studies, and future perspectives. The potential of Sindbis virus-based therapies for ovarian cancer treatment holds great promise and warrants further investigation. Investigations using other oncolytic viruses in preclinical studies and clinical trials are also presented. Full article

Alphaviruses, belonging to the Togaviridae family, and bunyaviruses, belonging to the Paramyxoviridae family, are globally distributed and lack FDA-approved vaccines and therapeutics. The alphaviruses Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV) are known to cause severe encephalitis, whereas Sindbis virus (SINV) causes arthralgia potentially persisting for years after initial infection. The bunyavirus Rift Valley Fever virus (RVFV) can lead to blindness, liver failure, and hemorrhagic fever. Brilacidin, a small molecule that was designed de novo based on naturally occurring host defensins, was investigated for its antiviral activity against these viruses in human small airway epithelial cells (HSAECs) and African green monkey kidney cells (Veros). This testing was further expanded into a non-enveloped Echovirus, a Picornavirus, to further demonstrate brilacidin’s effect on early steps of the viral infectious cycle that leads to inhibition of viral load. Brilacidin demonstrated antiviral activity against alphaviruses VEEV TC-83, VEEV TrD, SINV, EEEV, and bunyavirus RVFV. The inhibitory potential of brilacidin against the viruses tested in this study was dependent on the dosing strategy which necessitated compound addition pre- and post-infection, with addition only at the post-infection stage not eliciting a robust inhibitory response. The inhibitory activity of brilacidin was only modest in the context of the non-enveloped Picornavirus Echovirus, suggesting brilacidin may be less potent against non-enveloped viruses. Full article