Structure of Semliki Forest virus in complex with its receptor VLDLR.

Semliki Forest virus (SFV) is an alphavirus that uses the very-low-density lipoprotein receptor (VLDLR) as a receptor during infection of its vertebrate hosts and insect vectors. Herein, we used cryoelectron microscopy to study the structure of SFV in complex with VLDLR. We found that VLDLR binds multiple E1-DIII sites of SFV through its membrane-distal LDLR class A (LA) repeats. Among the LA repeats of the VLDLR, LA3 has the best binding affinity to SFV. The high-resolution structure shows that LA3 binds SFV E1-DIII through a small surface area of 378 Å2, with the main interactions at the interface involving salt bridges. Compared with the binding of single LA3s, consecutive LA repeats around LA3 promote synergistic binding to SFV, during which the LAs undergo a rotation, allowing simultaneous key interactions at multiple E1-DIII sites on the virion and enabling the binding of VLDLRs from divergent host species to SFV.

A novel three-plasmid packaging system for chimeric SFV/SIN VRPs derived from Semliki Forest virus and Sindbis virus as a candidate gene delivery vector.

Semliki Forest virus (SFV) viral replicon particles (VRPs) have been frequently used in various animal models and clinical trials. Chimeric replicon particles offer different advantages because of their unique biological properties. We here constructed a novel three-plasmid packaging system for chimeric SFV/SIN VRPs. The capsid and envelope of SIN structural proteins were generated using two-helper plasmids separately, and the SFV replicon contained the SFV replicase gene, packaging signal of SIN, subgenomic promoter followed by the exogenous gene, and 3' UTR of SIN. The chimeric VRPs carried luciferase or eGFP as reporter genes. The fluorescence and electron microscopy results revealed that chimeric VRPs were successfully packaged. The yield of the purified chimeric VRPs was approximately 2.5 times that of the SFV VRPs (1.38 × 107 TU/ml vs. 5.41 × 106 TU/ml) (p < 0.01). Furthermore, chimeric VRPs could be stored stably at 4°C for at least 60 days. Animal experiments revealed that mice immunized with chimeric VRPs (luciferase) had stronger luciferase expression than those immunized with equivalent amount of SFV VRPs (luciferase) (p < 0.01), and successfully expressed luciferase for approximately 12 days. Additionally, the chimeric VRPs expressed the RBD of SARS-CoV-2 efficiently and induced robust RBD-specific antibody responses in mice. In conclusion, the chimeric VRPs constructed here met the requirements of a gene delivery tool for vaccine development and cancer therapy.

Semliki Forest Virus (SFV) Self-Amplifying RNA Delivered to J774A.1 Macrophage Lineage by Its Association with a Purified Recombinant SFV Capsid Protein.

The Semliki Forest virus capsid protein (C) is an RNA binding protein which exhibits both specific and unspecific affinities to single-strand nucleic acids. The putative use of the self-amplifying RNAs (saRNAs) of alphaviruses for biotechnological purpose is one of the main studied strategies concerning RNA-based therapies or immunization. In this work, a recombinant C protein from SFV was expressed and purified from bacteria and used to associate in vitro with a saRNA derived from SFV. Results showed that the purified form of C protein can associate with the saRNA even after high temperature treatment. The C protein was associated with a modified saRNA coding for the green fluorescent protein (GFP) and delivered to murine macrophage cells which expressed the GFP, showing that the saRNA was functional after being associated with the recombinant purified C protein.

In Semliki Forest virus encephalitis, suppressor of cytokine signaling 4 (SOCS4) is an essential modulator of immune responses that mediates the balance between immunopathology and virus clearance.

Central nervous system virus infections are a major cause of morbidity and mortality worldwide and a significant global public health concern. As in many tissues, inflammation and immune responses in the brain, despite their protective roles, can also be harmful. Control of brain inflammation is important in many neurological diseases from encephalitis to multiple sclerosis and neurogenerative disease. The suppressors of cytokine signaling (SOCS) proteins are a key mechanism controlling inflammatory and immune responses across all tissues including the brain. Using a mouse model system, we demonstrate that lack of SOCS4 results in changes in the pathogenesis and clinical outcome of a neurotropic virus infection. Relative to wild-type mice, SOCS4-deficient mice showed accelerated clearance of virus from the brain, lower levels of persisting viral RNA in the brain, increased neuroinflammation and more severe neuropathology. We conclude that, in the mouse brain, SOCS4 is a vital regulator of antiviral immunity that mediates the critical balance between immunopathology and virus persistence.

Virus-Like Vesicles Based on Semliki Forest Virus-Containing Rabies Virus Glycoprotein Make a Safe and Efficacious Rabies Vaccine Candidate in a Mouse Model.

Rabies is a fatal zoonosis that causes encephalitis in mammals, and vaccination is the most effective method to control and eliminate rabies. Virus-like vesicles (VLVs), which are characterized as infectious, self-propagating membrane-enveloped particles composed of only Semliki Forest virus (SFV) replicase and vesicular stomatitis virus glycoprotein (VSV-G), have been proven safe and efficient as vaccine candidates. However, previous studies showed that VLVs containing rabies virus glycoprotein (RABV-G) grew at relatively low titers in cells, impeding their potential use as a rabies vaccine. In this study, we constructed novel VLVs by transfection of a mutant SFV RNA replicon encoding RABV-G. We found that these VLVs could self-propagate efficiently in cell culture and could evolve to high titers (approximately 108 focus-forming units [FFU]/ml) by extensive passaging 25 times in BHK-21 cells. Furthermore, we found that the evolved amino acid changes in SFV nonstructural protein 1 (nsP1) at positions 470 and 482 was critical for this high-titer phenotype. Remarkably, VLVs could induce robust type I interferon (IFN) expression in BV2 cells and were highly sensitive to IFN-α. We found that direct inoculation of VLVs into the mouse brain caused reduced body weight loss, mortality, and neuroinflammation compared with the RABV vaccine strain. Finally, it could induce increased generation of germinal center (GC) B cells, plasma cells (PCs), and virus-neutralizing antibodies (VNAs), as well as provide protection against virulent RABV challenge in immunized mice. This study demonstrated that VLVs containing RABV-G could proliferate in cells and were highly evolvable, revealing the feasibility of developing an economic, safe, and efficacious rabies vaccine. IMPORTANCE VLVs have been shown to represent a more versatile and superior vaccine platform. In previous studies, VLVs containing the Semliki Forest virus replicase (SFV nsP1 to nsP4) and rabies virus glycoprotein (RABV-G) grew to relatively low titers in cells. In our study, we not only succeeded in generating VLVs that proliferate in cells and stably express RABV-G, but the VLVs that evolved grew to higher titers, reaching 108 FFU/ml. We also found that nucleic acid changes at positions 470 and 482 in nsP1 were vital for this high-titer phenotype. Moreover, the VLVs that evolved in our studies were highly attenuated in mice, induced potent immunity, and protected mice from lethal RABV infection. Collectively, our study showed that high titers of VLVs containing RABV-G were achieved, demonstrating that these VLVs could be an economical, safe, and efficacious rabies vaccine candidate.

First-in-Human Phase I Clinical Trial of an SFV-Based RNA Replicon Cancer Vaccine against HPV-Induced Cancers.

A first-in-human phase I trial of Vvax001, an alphavirus-based therapeutic cancer vaccine against human papillomavirus (HPV)-induced cancers was performed assessing immunological activity, safety, and tolerability. Vvax001 consists of replication-incompetent Semliki Forest virus replicon particles encoding HPV16-derived antigens E6 and E7. Twelve participants with a history of cervical intraepithelial neoplasia were included. Four cohorts of three participants were treated per dose level, ranging from 5 × 105 to 2.5 × 108 infectious particles per immunization. The participants received three immunizations with a 3-week interval. For immune monitoring, blood was drawn before immunization and 1 week after the second and third immunization. Immunization with Vvax001 was safe and well tolerated, with only mild injection site reactions, and resulted in both CD4+ and CD8+ T cell responses against E6 and E7 antigens. Even the lowest dose of 5 × 105 infectious particles elicited E6/E7-specific interferon (IFN)-γ responses in all three participants in this cohort. Overall, immunization resulted in positive vaccine-induced immune responses in 12 of 12 participants in one or more assays performed. In conclusion, Vvax001 was safe and induced immune responses in all participants. These data strongly support further clinical evaluation of Vvax001 as a therapeutic vaccine in patients with HPV-related malignancies.

The Capsid Protein of Semliki Forest Virus Antagonizes RNA Interference in Mammalian Cells.

RNA interference (RNAi) is a conserved antiviral immune defense in eukaryotes, and numerous viruses have been found to encode viral suppressors of RNAi (VSRs) to counteract antiviral RNAi. Alphaviruses are a large group of positive-stranded RNA viruses that maintain their transmission and life cycles in both mosquitoes and mammals. However, there is little knowledge about how alphaviruses antagonize RNAi in both host organisms. In this study, we identified that Semliki Forest virus (SFV) capsid protein can efficiently suppress RNAi in both insect and mammalian cells by sequestrating double-stranded RNA and small interfering RNA. More importantly, when the VSR activity of SFV capsid was inactivated by reverse genetics, the resulting VSR-deficient SFV mutant showed severe replication defects in mammalian cells, which could be rescued by blocking the RNAi pathway. Besides, capsid protein of Sindbis virus also inhibited RNAi in cells. Together, our findings show that SFV uses capsid protein as VSR to antagonize RNAi in infected mammalian cells, and this mechanism is probably used by other alphaviruses, which shed new light on the knowledge of SFV and alphavirus.IMPORTANCE Alphaviruses are a genus of positive-stranded RNA viruses and include numerous important human pathogens, such as Chikungunya virus, Ross River virus, Western equine encephalitis virus, etc., which create the emerging and reemerging public health threat worldwide. RNA interference (RNAi) is one of the most important antiviral mechanisms in plants and insects. Accumulating evidence has provided strong support for the existence of antiviral RNAi in mammals. In response to antiviral RNAi, viruses have evolved to encode viral suppressors of RNAi (VSRs) to antagonize the RNAi pathway. It is unclear whether alphaviruses encode VSRs that can suppress antiviral RNAi during their infection in mammals. In this study, we first uncovered that capsid protein encoded by Semliki Forest virus (SFV), a prototypic alphavirus, had a potent VSR activity that can antagonize antiviral RNAi in the context of SFV infection in mammalian cells, and this mechanism is probably used by other alphaviruses.

Herpes Simplex Virus 1 Can Enter Dynamin 1 and 2 Double-Knockout Fibroblasts.

Dynamin GTPases, best known for their role in membrane fission of endocytic vesicles, provide a target for viruses to be exploited during endocytic uptake. Recently, we found that entry of herpes simplex virus 1 (HSV-1) into skin cells depends on dynamin, although our results supported that viral internalization occurs via both direct fusion with the plasma membrane and via endocytic pathways. To further explore the role of dynamin for efficient HSV-1 entry, we utilized conditional dynamin 1 and dynamin 2 double-knockout (DKO) fibroblasts as an experimental tool. Strikingly, HSV-1 entered control and DKO fibroblasts with comparable efficiencies. For comparison, we infected DKO cells with Semliki Forest virus, which is known to adopt clathrin-mediated endocytosis as its internalization pathway, and observed efficient virus entry. These results support the notion that the DKO cells provide alternative pathways for viral uptake. Treatment of cells with the dynamin inhibitor dynasore confirmed that HSV-1 entry depended on dynamin in the control fibroblasts. As expected, dynasore did not interfere with viral entry into DKO cells. Electron microscopy of HSV-1-infected cells suggests viral entry after fusion with the plasma membrane and by endocytosis in both dynamin-expressing and dynamin-deficient cells. Infection at low temperatures where endocytosis is blocked still resulted in HSV-1 entry, although at a reduced level, which suggests that nonendocytic pathways contribute to successful entry. Overall, our results strengthen the impact of dynamin for HSV-1 entry, as only cells that adapt to the lack of dynamin allow dynamin-independent entry.IMPORTANCE The human pathogen herpes simplex virus 1 (HSV-1) can adapt to a variety of cellular pathways to enter cells. In general, HSV-1 is internalized by fusion of its envelope with the plasma membrane or by endocytic pathways, which reflects the high adaptation to differences in its target cells. The challenges are to distinguish whether multiple or only one of these internalization pathways leads to successful entry and, furthermore, to identify the mode of viral uptake. In this study, we focused on dynamin, which promotes endocytic vesicle fission, and explored how the presence and absence of dynamin can influence viral entry. Our results support the idea that HSV-1 entry into mouse embryonic fibroblasts depends on dynamin; however, depletion of dynamin still allows efficient viral entry, suggesting that alternative pathways present upon dynamin depletion can accomplish viral internalization.

An alphavirus-based therapeutic cancer vaccine: from design to clinical trial.

Cancer immunotherapy has greatly advanced in recent years. Most immunotherapeutic strategies are based on the use of immune checkpoint blockade to unleash antitumor immune responses or on the induction or adoptive transfer of immune effector cells. We aim to develop therapeutic vaccines based on recombinant Semliki Forest virus vectors to induce tumor-specific effector immune cells. In this review, we describe our ongoing work on SFV-based vaccines targeted against human papillomavirus- and hepatitis C virus-related infections and malignancies, focusing on design, delivery, combination strategies, preclinical efficacy and product development for a first-in-man clinical trial with an HPV-specific vaccine.

Purification of Highly Active Alphavirus Replication Complexes Demonstrates Altered Fractionation of Multiple Cellular Membranes.

Positive-strand RNA viruses replicate their genomes in membrane-associated structures; alphaviruses and many other groups induce membrane invaginations called spherules. Here, we established a protocol to purify these membranous replication complexes (RCs) from cells infected with Semliki Forest virus (SFV). We isolated SFV spherules located on the plasma membrane and further purified them using two consecutive density gradients. This revealed that SFV infection strongly modifies cellular membranes. We removed soluble proteins, the Golgi membranes, and most of the mitochondria, but plasma membrane, endoplasmic reticulum (ER), and late endosome markers were retained in the membrane fraction that contained viral RNA synthesizing activity, replicase proteins, and minus- and plus-strand RNA. Electron microscopy revealed that the purified membranes displayed spherule-like structures with a narrow neck. This membrane enrichment was specific to viral replication, as such a distribution of membrane markers was only observed after infection. Besides the plasma membrane, SFV infection remodeled the ER, and the cofractionation of the RC-carrying plasma membrane and ER suggests that SFV recruits ER proteins or membrane to the site of replication. The purified RCs were highly active in synthesizing both genomic and subgenomic RNA. Detergent solubilization destroyed the replication activity, demonstrating that the membrane association of the complex is essential. Most of the newly made RNA was in double-stranded replicative molecules, but the purified complexes also produced single-stranded RNA as well as released newly made RNA. This indicates that the purification established here maintained the functionality of RCs and thus enables further structural and functional studies of active RCs.IMPORTANCE Similar to all positive-strand RNA viruses, the arthropod-borne alphaviruses induce membranous genome factories, but little is known about the arrangement of viral replicase proteins and the presence of host proteins in these replication complexes. To improve our knowledge of alphavirus RNA-synthesizing complexes, we isolated and purified them from infected mammalian cells. Detection of viral RNA and in vitro replication assays revealed that these complexes are abundant and highly active when located on the plasma membrane. After multiple purification steps, they remain functional in synthesizing and releasing viral RNA. Besides the plasma membrane, markers for the endoplasmic reticulum and late endosomes were enriched with the replication complexes, demonstrating that alphavirus infection modified cellular membranes beyond inducing replication spherules on the plasma membrane. We have developed here a gentle purification method to obtain large quantities of highly active replication complexes, and similar methods can be applied to other positive-strand RNA viruses.

Alphavirus Restriction by IFITM Proteins.

Interferon inducible transmembrane proteins (IFITMs) are broad-spectrum antiviral factors. In cell culture the entry of many enveloped viruses, including orthomyxo-, flavi-, and filoviruses, is inhibited by IFITMs, though the mechanism(s) involved remain unclear and may vary between viruses. We demonstrate that Sindbis and Semliki Forest virus (SFV), which both use endocytosis and acid-induced membrane fusion in early endosomes to infect cells, are restricted by the early endosomal IFITM3. The late endosomal IFITM2 is less restrictive and the plasma membrane IFITM1 does not inhibit normal infection by either virus. IFITM3 inhibits release of the SFV capsid into the cytosol, without inhibiting binding, internalization, trafficking to endosomes or low pH-induced conformational changes in the envelope glycoprotein. Infection by SFV fusion at the cell surface was inhibited by IFITM1, but was equally inhibited by IFITM3. Furthermore, an IFITM3 mutant (Y20A) that is localized to the plasma membrane inhibited infection by cell surface fusion more potently than IFITM1. Together, these results indicate that IFITMs, in particular IFITM3, can restrict alphavirus infection by inhibiting viral fusion with cellular membranes. That IFITM3 can restrict SFV infection by fusion at the cell surface equivalently to IFITM1 suggests that IFITM3 has greater antiviral potency against SFV.

Optogenetic manipulation of medullary neurons in the locust optic lobe.

The locust is a widely used animal model for studying sensory processing and its relation to behavior. Due to the lack of genomic information, genetic tools to manipulate neural circuits in locusts are not yet available. We examined whether Semliki Forest virus is suitable to mediate exogenous gene expression in neurons of the locust optic lobe. We subcloned a channelrhodopsin variant and the yellow fluorescent protein Venus into a Semliki Forest virus vector and injected the virus into the optic lobe of locusts ( Schistocerca americana). Fluorescence was observed in all injected optic lobes. Most neurons that expressed the recombinant proteins were located in the first two neuropils of the optic lobe, the lamina and medulla. Extracellular recordings demonstrated that laser illumination increased the firing rate of medullary neurons expressing channelrhodopsin. The optogenetic activation of the medullary neurons also triggered excitatory postsynaptic potentials and firing of a postsynaptic, looming-sensitive neuron, the lobula giant movement detector. These results indicate that Semliki Forest virus is efficient at mediating transient exogenous gene expression and provides a tool to manipulate neural circuits in the locust nervous system and likely other insects. NEW & NOTEWORTHY Using Semliki Forest virus, we efficiently delivered channelrhodopsin into neurons of the locust optic lobe. We demonstrate that laser illumination increases the firing of the medullary neurons expressing channelrhodopsin and elicits excitatory postsynaptic potentials and spiking in an identified postsynaptic target neuron, the lobula giant movement detector neuron. This technique allows the manipulation of neuronal activity in locust neural circuits using optogenetics.

Partially Uncleaved Alphavirus Replicase Forms Spherule Structures in the Presence and Absence of RNA Template.

Alphaviruses are positive-strand RNA viruses expressing their replicase as a polyprotein, P1234, which is cleaved to four final products, nonstructural proteins nsP1 to nsP4. The replicase proteins together with viral RNA and host factors form membrane invaginations termed spherules, which act as the replication complexes producing progeny RNAs. We have previously shown that the wild-type alphavirus replicase requires a functional RNA template and active polymerase to generate spherule structures. However, we now find that specific partially processed forms of the replicase proteins alone can give rise to membrane invaginations in the absence of RNA or replication. The minimal requirement for spherule formation was the expression of properly cleaved nsP4, together with either uncleaved P123 or with the combination of nsP1 and uncleaved P23. These inactive spherules were morphologically less regular than replication-induced spherules. In the presence of template, nsP1 plus uncleaved P23 plus nsP4 could efficiently assemble active replication spherules producing both negative-sense and positive-sense RNA strands. P23 alone did not have membrane affinity, but could be recruited to membrane sites in the presence of nsP1 and nsP4. These results define the set of viral components required for alphavirus replication complex assembly and suggest the possibility that it could be reconstituted from separately expressed nonstructural proteins.IMPORTANCE All positive-strand RNA viruses extensively modify host cell membranes to serve as efficient platforms for viral RNA replication. Alphaviruses and several other groups induce protective membrane invaginations (spherules) as their genome factories. Most positive-strand viruses produce their replicase as a polyprotein precursor, which is further processed through precise and regulated cleavages. We show here that specific cleavage intermediates of the alphavirus replicase can give rise to spherule structures in the absence of viral RNA. In the presence of template RNA, the same intermediates yield active replication complexes. Thus, partially cleaved replicase proteins play key roles that connect replication complex assembly, membrane deformation, and the different stages of RNA synthesis.

Pseudo-typed Semliki Forest virus delivers EGFP into neurons.

Semliki Forest virus (SFV), a neurotropic virus, has been used to deliver heterologous genes into cells in vitro and in vivo. In this study, we constructed a reporter SFV4-FL-EGFP and found that it can deliver EGFP into neurons located at the injection site without disseminating throughout the brain. Lacking of the capsid gene of SFV4-FL-EGFP does not block its life cycle, while forming replication-competent virus-like particles (VLPs). These VLPs hold subviral genome by using the packaging sequence (PS) located within the nsP2 gene, and can transfer their genome into cells. In addition, we found that the G protein of vesicular stomatitis virus (VSVG) can package SFV subviral genome, which is consistent with the previous reports. The G protein of rabies virus (RVG) could also package SFV subviral genome. These pseudo-typed SFV can deliver EGFP gene into neurons. Taken together, these findings may be used to construct various SFV-based delivery systems for virological studies, gene therapy, and neural circuit labeling.

Capsid-deficient alphaviruses generate propagative infectious microvesicles at the plasma membrane.

Alphavirus budding is driven by interactions between nucleocapsids assembled in the cytoplasm and envelope proteins present at the plasma membrane. So far, the expression of capsid and envelope proteins in infected cells has been considered an absolute requirement for alphavirus budding and propagation. In the present study, we show that Semliki Forest virus and Sindbis virus lacking the capsid gene can propagate in mammalian and insect cells. This propagation is mediated by the release of infectious microvesicles (iMVs), which are pleomorphic and have a larger size and density than wild-type virus. iMVs, which contain viral RNA inside and viral envelope proteins on their surface, are released at the plasma membrane and infect cells using the endocytic pathway in a similar way to wild-type virus. iMVs are not pathogenic in immunocompetent mice when injected intravenously, but can infect different organs like lungs and heart. Finally, we also show that alphavirus genomes without capsid can mediate the propagation of heterologous genes, making these vectors potentially interesting for gene therapy or vaccination studies. The minimalist infectious system described in this study shows that a self-replicating RNA able to express membrane proteins with binding and fusion properties is able to propagate, providing some insights into virus evolution.

Therapeutic immunization and local low-dose tumor irradiation, a reinforcing combination.

Therapeutic cancer vaccines show promise in preclinical studies, yet their clinical efficacy is limited. Increased recruitment of immune cells into tumors and suppression of the immune suppressive tumor environment are critical components toward effective cancer immunotherapies. Here, we report how local low-dose irradiation, alone or with a therapeutic immunization based on Semliki Forest virus (SFV) against human papillomavirus (HPV)-related cancer, influences these immune mechanisms. We first demonstrated that immunization with SFVeE6,7 or SFVeOVA, replicon particles expressing either HPV16 E6/E7 or ovalbumin, resulted in an antigen-specific migration of CD8+ T cells into HPV- and OVA-specific tumors. Local low-dose tumor irradiation alone resulted in a 2-fold increase of intratumoral CD8+ T cells. When 14 Gy irradiation was combined with immunization, intratumoral numbers of CD8+ T cells increased 10-fold and the number of CD8+ T cells specific for the E7- epitope increased more than 20-fold. Irradiation alone however also increased the number of intratumoral myeloid-derived suppressor cells (MDSCs) 3.5-fold. Importantly, this number did not further increase when combined with immunization. As a result, the ratio of antigen-specific CD8+ T cells and MDSCs in tumors increased up to 85-fold compared to the control. We furthermore demonstrated that following irradiation CCR2 and CCL2, CXCR6 and CCL16, chemokines and ligands involved in tumor homing of immune cells, were significantly up regulated. This study demonstrates that local low-dose tumor irradiation influences the intratumoral immune population induced by SFVeE6,7 immunization by a strong increase in the ratio of antitumoral to immune suppressive cells, thus changing the intratumoral immune balance in favor of antitumor activity.

Gene silencing in the aedine cell lines C6/36 and U4.4 using long double-stranded RNA.

BACKGROUND: RNA interference (RNAi) is a target-specific gene silencing method that can be used to determine gene functions and investigate host-pathogen interactions, as well as facilitating the development of ecofriendly pesticides. Commercially available transfection reagents (TRs) can improve the efficacy of RNAi. However, we currently lack a product and protocol for the transfection of insect cell lines with long double-stranded RNA (dsRNA). METHODS: We used agarose gel electrophoresis to determine the capacity of eight TRs to form complexes with long dsRNA. A CellTiter-Glo assay was then used to assess the cytotoxicity of the resulting lipoplexes. We also measured the cellular uptake of dsRNA by fluorescence microscopy using the fluorophore Cy3 as a label. Finally, we analyzed the TRs based on their transfection efficacy and compared the RNAi responses of Aedes albopictus C6/36 and U4.4 cells by knocking down an mCherry reporter Semliki Forest virus in both cell lines. RESULTS: The TRs from Biontex (K4, Metafectene Pro, and Metafectene SI+) showed the best complexing capacity and the lowest dsRNA:TR ratio needed for complete complex formation. Only HiPerFect was unable to complex the dsRNA completely, even at a ratio of 1:9. Most of the complexes containing mCherry-dsRNA were nontoxic at 2 ng/µL, but Lipofectamine 2000 was toxic at 1 ng/µL in U4.4 cells and at 2 ng/µL in C6/36 cells. The transfection of U4.4 cells with mCherry-dsRNA/TR complexes achieved significant knockdown of the virus reporter. Comparison of the RNAi response in C6/36 and U4.4 cells suggested that C6/36 cells lack the antiviral RNAi response because there was no significant knockdown of the virus reporter in any of the treatments. CONCLUSIONS: C6/36 cells have an impaired RNAi response as previously reported. This investigation provides valuable information for future RNAi experiments by showing how to mitigate the adverse effects attributed to TRs. This will facilitate the judicious selection of TRs and transfection conditions conducive to RNAi research in mosquitoes.

Analyzing the factors affecting virus invasion by quantitative single-particle analysis.

Viral diseases are among the main threats to public health. Understanding the factors affecting viral invasion is important for antiviral research. Until now, it was known that most viruses have very low plaque-forming unit (PFU)-to-particle ratios. However, further investigation is required to determine the underlying factors. Here, using quantitative single-particle analysis methods, the invasion of Semliki Forest virus (SFV), Japanese encephalitis virus (JEV), and influenza A virus (IAV) containing attachment to the cell surface, entry into the cell, transport towards the cell interior, and fusion with endosomes to release nucleocapsids were quantitatively analysed in parallel. It was found that for SFV with an PFU-to-particle ratio of approximately 1:2, an entry efficiency of approximately 31% limited infection. For JEV, whose PFU-to-particle ratio was approximately 1:310, an attachment efficiency of approximately 27% and an entry efficiency of 10% were the main factors limiting its infection. Meanwhile, for IAV with PFU-to-particle ratios of 1:8100, 5% attachment efficiency, 9% entry efficiency, and 53% fusion efficiency significantly limited its infection. These results suggest that viruses with different infectivities have different limited steps in the invasion process. Moreover, there are significant differences in attachment efficiencies among viruses, emphasizing the pivotal role of attachment in viral invasion. The influence of the virus purification method on virus invasion was also investigated. This study, for the first time, reports the efficiencies of different stages of virus invasion, leading to a better understanding of virus invasion and providing a protocol to quantitatively analyse the virus invasion efficiency.

A high throughput antiviral screening platform for alphaviruses based on Semliki Forest virus expressing eGFP reporter gene.

Alphaviruses, which contain a variety of mosquito-borne pathogens, are important pathogens of emerging/re-emerging infectious diseases and potential biological weapons. Currently, no specific antiviral drugs are available for the treatment of alphaviruses infection. For most highly pathogenic alphaviruses are classified as risk group-3 agents, the requirement of biosafety level 3 (BSL-3) facilities limits the live virus-based antiviral study. To facilitate the antiviral development of alphaviruses, we developed a high throughput screening (HTS) platform based on a recombinant Semliki Forest virus (SFV) which can be manipulated in BSL-2 laboratory. Using the reverse genetics approach, the recombinant SFV and SFV reporter virus expressing eGFP (SFV-eGFP) were successfully rescued. The SFV-eGFP reporter virus exhibited robust eGFP expression and remained relatively stable after four passages in BHK-21 â€‹cells. Using a broad-spectrum alphavirus inhibitor ribavirin, we demonstrated that the SFV-eGFP can be used as an effective tool for antiviral study. The SFV-eGFP reporter virus-based HTS assay in a 96-well format was then established and optimized with a robust Z' score. A section of reference compounds that inhibit highly pathogenic alphaviruses were used to validate that the SFV-eGFP reporter virus-based HTS assay enables rapid screening of potent broad-spectrum inhibitors of alphaviruses. This assay provides a safe and convenient platform for antiviral study of alphaviruses.

Alphavirus infection triggers antiviral RNAi immunity in mammals.

RNA interference (RNAi) is a well-established antiviral immunity. However, for mammalian somatic cells, antiviral RNAi becomes evident only when viral suppressors of RNAi (VSRs) are disabled by mutations or VSR-targeting drugs, thereby limiting its scope as a mammalian immunity. We find that a wild-type alphavirus, Semliki Forest virus (SFV), triggers the Dicer-dependent production of virus-derived small interfering RNAs (vsiRNAs) in both mammalian somatic cells and adult mice. These SFV-vsiRNAs are located at a particular region within the 5' terminus of the SFV genome, Argonaute loaded, and active in conferring effective anti-SFV activity. Sindbis virus, another alphavirus, also induces vsiRNA production in mammalian somatic cells. Moreover, treatment with enoxacin, an RNAi enhancer, inhibits SFV replication dependent on RNAi response in vitro and in vivo and protects mice from SFV-induced neuropathogenesis and lethality. These findings show that alphaviruses trigger the production of active vsiRNA in mammalian somatic cells, highlighting the functional importance and therapeutic potential of antiviral RNAi in mammals.