Search Results (36)

Mechanisms of arboviral neuroinvasion are still incompletely resolved, despite longstanding emphasis on the blood-brain barrier (BBB) as the principal interface for central nervous system (CNS) entry. While BBB-centered models have been highly informative, they may underrepresent the contribution of other CNS border structures, particularly the choroid plexus and the blood-cerebrospinal fluid barrier (BCSFB). Here, we re-examine the BCSFB as a relevant but unevenly supported neuroinvasion interface in arboviral encephalitis. The strongest direct evidence is currently available for Zika virus (ZIKV), for which experimental studies support infection of choroid plexus-associated cells and CNS access through the blood-CSF axis. Semliki Forest virus (SFV) provides additional direct, although still limited, support for this concept. In contrast, for West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV), evidence for choroid plexus involvement remains indirect or insufficiently resolved, even though neuroinvasion itself is well established. We therefore argue not for replacement of BBB-centered models, but for broader integration of the BCSFB into current frameworks of arboviral CNS invasion. This evidence-based perspective supports a hierarchical, virus-dependent view of choroid plexus involvement and highlights the need for mechanistic studies that directly test when and how this interface contributes to encephalitic disease. Full article

Background/Objectives: Rabies is a fatal zoonotic disease caused by the rabies virus (RABV), and effective therapeutic treatments are currently lacking. Vaccination remains the primary strategy for rabies control. The Semliki Forest virus-rabies virus glycoprotein (SFV-RVG), a virus-like vesicle rabies vaccine combining Semliki Forest virus replicase and rabies glycoprotein, has shown potential as a promising vaccine candidate. This study aimed to optimize the production of SFV-RVG and evaluate adjuvant formulations to improve its immunogenicity in both mice and dogs. Methods: SFV-RVG production was optimized by determining the optimal multiplicity of infection (MOI) at 0.03 and cell density at 1 × 10⁶–1.3 × 10⁶ cells/mL, followed by scaling up the process in bioreactors. Eleven adjuvant formulations were tested in mice and dogs to assess their effects on immunogenicity. Cytokine analysis and antibody responses were measured, including IFN-γ, IL-4, IgG2a/IgG1 ratios, and neutralizing antibody titers. Results: The optimized SFV-RVG production was successfully scaled up, and M103 adjuvant induced rapid early antibody titers in mice. In dogs, GEL02 led to the highest neutralizing antibody levels, exceeding 40 IU/mL by 28 days post-immunization. Cytokine analysis indicated that both M103 and GEL02 significantly enhanced IFN-γ and IL-4 expression, balancing the Th1/Th2 immune response. SFV-RVG with GEL02 demonstrated stronger immunogenicity than a commercial vaccine, and challenge studies confirmed robust protection against lethal RABV in mice. Conclusions: This study establishes GEL02 as a superior adjuvant for rabies vaccines and provides a scalable SFV-RVG production process. These findings highlight SFV-RVG with GEL02 as a promising rabies vaccine candidate for dogs, offering significant potential for rabies control. Full article

Ticks are important vectors of a wide range of pathogens with significant medical and veterinary importance. Different tick species occupy different habitats with an overall widespread geographical distribution. In addition to their role as reservoirs or vectors, ticks are involved in maintaining pathogens in the environment and among wild and domestic animals. In this study, tick species infesting wild animals, as well as collected from the environment and their pathogens reported in 17 countries in Africa between 2003 and 2023, were collected according to the PRISMA guidelines. Data on ticks resulted in a total of 40 different tick species from 35 different wild animal species. Among the ticks, 34 infectious agents were noted including parasitic (Babesia, Theileria, Hepatozoon, Eimeria), bacterial (Anaplasma, Bartonella, Borrelia, Candidatus Midichloria mitochondrii, Candidatus Allocryptoplasma spp., Coxiella, Ehrlichia, Francisella, and Rickettsia), and a surprisingly high diversity of viral pathogens (Bunyamwera virus, Crimean-Congo Haemorhagic Fever virus, Ndumu virus, Semliki Forest virus, Thogoto virus, West Nile virus). These results highlight the public health and veterinary importance of the information on tick-borne infections. This knowledge is essential to strive to implement programs for sustainable control of ticks and tick-borne diseases. Full article

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. Full article

The intracellular bacterium Wolbachia is increasingly being utilised in control programs to limit the spread of arboviruses by Aedes mosquitoes. Achieving a better understanding of how Wolbachia strains can reduce viral replication/spread could be important for the long-term success of such programs. Previous studies have indicated that for some strains of Wolbachia, perturbations in lipid metabolism and cholesterol storage are vital in Wolbachia-mediated antiviral activity against the flaviviruses dengue and Zika; however, it has not yet been examined whether arboviruses in the alphavirus group are affected in the same way. Here, using the reporters for the alphavirus Semliki Forest virus (SFV) in Aedes albopictus cells, we found that Wolbachia strains wMel, wAu and wAlbB blocked viral replication/translation early in infection and that storage of cholesterol in lipid droplets is not key to this inhibition. Another alphavirus, o’nyong nyong virus (ONNV), was tested in both Aedes albopictus cells and in vivo in stable, transinfected Aedes aegypti mosquito lines. The strains wMel, wAu and wAlbB show strong antiviral activity against ONNV both in vitro and in vivo. Again, 2-hydroxypropyl-β-cyclodextrin (2HPCD) was not able to rescue ONNV replication in cell lines, suggesting that the release of stored cholesterol caused by wMel is not able to rescue blockage of ONNV. Taken together, this study shows that alphaviruses appear to be inhibited early in replication/translation and that there may be differences in how alphaviruses are inhibited by Wolbachia in comparison to flaviviruses. Full article

The development and testing of antimicrobial coatings continues to be a crucial approach, considering the ongoing emergence of antibiotic-resistant bacteria and the rapid transmission of highly pathogenic viruses. In this study, three types of coatings—pure metallic copper (Cu), zinc oxide (ZnO), and a three-layer zinc oxide and copper mixed coating (ZnO/Cu/ZnO)—were deposited by magnetron sputtering on polyethylene terephthalate substrates to evaluate their antimicrobial potential using various microorganisms, including viruses. Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli bacteria were used for the assessment of antibacterial properties. Antiviral testing was performed using MS2 bacteriophage and replication-deficient Semliki Forest virus, both representing single-stranded RNA-containing viruses. The samples’ ability to cause reactive oxygen species formation was measured, and the effect on bacterial metabolic activity was evaluated. Cu-coated samples showed high inhibitory activity (>95%) against E. coli and S. aureus bacteria, as well as against tested viruses (SFV and MS2). The antibacterial and antiviral properties of ZnO/Cu/ZnO and ZnO coatings were not significant. Although ZnO/Cu/ZnO and ZnO caused inhibition of the metabolic activity of the bacteria, it was insufficient for complete bacteria eradication. Furthermore, significant reactive oxygen species (ROS) production was detected only for single Cu-coated samples, correlating with the strong bacteria-killing ability. We suppose that the ZnO layer exhibited a low release of Zn ions and prevented contact of the Cu layer with bacteria and viruses in the ZnO/Cu/ZnO coating. We conclude that current ZnO and Cu-ZnO-layered coatings do not possess antibacterial and antiviral activity. Full article

Sindbis alphavirus vectors offer a promising platform for cancer therapy, serving as valuable models for alphavirus-based treatment. This review emphasizes key studies that support the targeted delivery of Sindbis vectors to tumor cells, highlighting their effectiveness in expressing tumor-associated antigens and immunomodulating proteins. Among the various alphavirus vectors developed for cancer therapy, Sindbis-vector-based imaging studies have been particularly extensive. Imaging modalities that enable the in vivo localization of Sindbis vectors within lymph nodes and tumors are discussed. The correlation between laminin receptor expression, tumorigenesis, and Sindbis virus infection is examined. Additionally, we present alternative entry receptors for Sindbis and related alphaviruses, such as Semliki Forest virus and Venezuelan equine encephalitis virus. The review also discusses cancer treatments that are based on the alphavirus vector expression of anti-tumor agents, including tumor-associated antigens, cytokines, checkpoint inhibitors, and costimulatory immune molecules. Full article

The presence of alphaviruses, such as chikungunya virus (CHIKV), has never been reported in Mauritania. We assessed the seroprevalence of CHIKV among Nouakchott residents. A cross-sectional study involving 1300 non-febrile patients consulting at the Nouakchott hospital center was conducted between January and June 2021. The presence of anti-CHIKV IgG and neutralizing antibodies against CHIKV, O’nyong-nyong virus (ONNV), and Semliki Forest virus (SFV) was determined by an enzyme-linked immunosorbent assay (ELISA) and a serum neutralization test, respectively, and the associated risk factors were investigated. Of the 1300 study participants, serological evidence of previous exposure to CHIKV was observed in 37 individuals (2.8%). Sex, age, reported use of repellants, and bed net ownership and usage were not associated with CHIKV seropositivity. Our results showed the co-circulation of two other alphaviruses, ONNV and SFV, in Nouakchott in 30 (2.3%) individuals. This is the first study that documents the co-circulation of CHIKV, ONNV, and SFV in Mauritania, albeit at low prevalence. Surveillance and routine testing for alphaviruses and other arboviruses in symptomatic patients should be implemented in health facilities to assess the health burden associated with these viruses. Efforts should also be made to strengthen the vector control measures. Full article

African swine fever virus (ASFV) causes a devastating viral hemorrhagic disease in domestic pigs and Eurasian wild boars, posing a foremost threat to the swine industry and pig farming. The development of an effective vaccine is urgently needed, but has been hampered by the lack of an in-depth, mechanistic understanding of the host immune response to ASFV infection and the induction of protective immunity. In this study, we report that immunization of pigs with Semliki Forest Virus (SFV) replicon-based vaccine candidates expressing ASFV p30, p54, and CD2v, as well as their ubiquitin-fused derivatives, elicits T cell differentiation and expansion, promoting specific T cell and humoral immunity. Due to significant variations in the individual non-inbred pigs in response to the vaccination, a personalized analysis was conducted. Using integrated analysis of differentially expressed genes (DEGs), Venn, KEGG and WGCNA, Toll-like receptor, C-type lectin receptor, IL17 receptor, NOD-like receptor and nucleic acid sensor-mediated signaling pathways were demonstrated to be positively correlated to the antigen-stimulated antibody production and inversely correlated to the IFN-γ secreting cell counts in peripheral blood mononuclear cells (PBMCs). An up-regulation of CIQA, CIQB, CIQC, C4BPA, SOSC3, S100A8 and S100A9, and down-regulation of CTLA4, CXCL2, CXCL8, FOS, RGS1, EGR1 and SNAI1 are general in the innate immune response post-the second boost. This study reveals that pattern recognition receptors TLR4, DHX58/DDX58 and ZBP1, and chemokines CXCL2, CXCL8 and CXCL10 may play important roles in regulating this vaccination-stimulated adaptive immune response. Full article

The quantification of viruses is necessary for both research and clinical applications. The methods available for RNA virus quantification possess several drawbacks, including sensitivity to inhibitors and the necessity of a standard curve generation. The main purpose of this study was to develop and validate a method for the quantification of recombinant, replication-deficient Semliki Forest virus (SFV) vectors using droplet digital PCR (ddPCR). This technique demonstrated stability and reproducibility using various sets of primers that targeted inserted transgenes, as well as the nsP1 and nsP4 genes of the SFV genome. Furthermore, the genome titers in the mixture of two types of replication-deficient recombinant virus particles were successfully measured after optimizing the annealing/extension temperature and virus:virus ratios. To measure the infectious units, we developed a single-cell ddPCR, adding the whole infected cells to the droplet PCR mixture. Cell distribution in the droplets was investigated, and β-actin primers were used to normalize the quantification. As a result, the number of infected cells and the virus infectious units were quantified. Potentially, the proposed single-cell ddPCR approach could be used to quantify infected cells for clinical applications. Full article

Mosquito-borne diseases caused by viruses and parasites are responsible for more than 700 million infections each year. Anopheles and Aedes are the two major vectors for, respectively, malaria and arboviruses. Anopheles mosquitoes are the primary vector of just one known arbovirus, the alphavirus o’nyong-nyong virus (ONNV), which is closely related to the chikungunya virus (CHIKV), vectored by Aedes mosquitoes. However, Anopheles harbor a complex natural virome of RNA viruses, and a number of pathogenic arboviruses have been isolated from Anopheles mosquitoes in nature. CHIKV and ONNV are in the same antigenic group, the Semliki Forest virus complex, are difficult to distinguish via immunodiagnostic assay, and symptomatically cause essentially the same human disease. The major difference between the arboviruses appears to be their differential use of mosquito vectors. The mechanisms governing this vector specificity are poorly understood. Here, we summarize intrinsic and extrinsic factors that could be associated with vector specificity by these viruses. We highlight the complexity and multifactorial aspect of vectorial specificity of the two alphaviruses, and evaluate the level of risk of vector shift by ONNV or CHIKV. Full article

Culex spp. mosquitoes are important vectors of viruses, such as West Nile virus, Eastern equine encephalitis virus and Rift valley fever virus. However, their interactions with innate antiviral immunity, especially RNA interference (RNAi), are not well known. Most research on RNAi pathways in mosquitoes is focused on the tropical vector mosquito Aedes aegypti. Here, we investigated the production of arbovirus-specific small RNAs in Cx. quinquefasciatus-derived HSU cells. Furthermore, by silencing RNAi-related proteins, we investigated the antiviral role of these proteins for two different arboviruses: Semliki Forest virus (SFV) and Bunyamwera orthobunyavirus (BUNV). Our results showed an expansion of Ago2 and Piwi6 in Cx. quinquefasciatus compared to Ae. aegypti. While silencing Ago2a and Ago2b increased BUNV replication, only Ago2b showed antiviral activity against SFV. Our results suggest differences in the function of Cx. quinquefasciatus and Ae. aegypti RNAi proteins and highlight the virus-specific function of these proteins in Cx. quinquefasciatus. Full article

Alphaviruses have been engineered as expression vectors for vaccine development and gene therapy. Due to the feature of RNA self-replication, alphaviruses can provide exceptional direct cytoplasmic expression of transgenes based on the delivery of recombinant particles, naked or nanoparticle-encapsulated RNA or plasmid-based DNA replicons. Alphavirus vectors have been utilized for the expression of various antigens targeting different types of cancers, and cytotoxic and antitumor genes. The most common alphavirus vectors are based on the Semliki Forest virus, Sindbis virus and Venezuelan equine encephalitis virus, but the oncolytic M1 alphavirus has also been used. Delivery of immunostimulatory cytokine genes has been the basis for immunotherapy demonstrating efficacy in different animal tumor models for brain, breast, cervical, colon, lung, ovarian, pancreatic, prostate and skin cancers. Typically, therapeutic effects including tumor regression, tumor eradication and complete cure as well as protection against tumor challenges have been observed. Alphavirus vectors have also been subjected to clinical evaluations. For example, therapeutic responses in all cervical cancer patients treated with an alphavirus vector expressing the human papilloma virus E6 and E7 envelope proteins have been achieved. Full article

Interferon gamma (IFNg) is a pleiotropic cytokine that can potentially reprogram the tumor microenvironment; however, the antitumor immunomodulatory properties of IFNg still need to be validated due to variable therapeutic outcomes in preclinical and clinical studies. We developed a replication-deficient Semliki Forest virus vector expressing IFNg (SFV/IFNg) and evaluated its immunomodulatory antitumor potential in vitro in a model of 3D spheroids and in vivo in an immunocompetent 4T1 mouse breast cancer model. We demonstrated that SFV-derived, IFN-g-stimulated bone marrow macrophages can be used to acquire the tumoricidal M1 phenotype in 3D nonattached conditions. Coculturing SFV/IFNg-infected 4T1 spheroids with BMDMs inhibited spheroid growth. In the orthotopic 4T1 mouse model, intratumoral administration of SFV/IFNg virus particles alone or in combination with the Pam3CSK4 TLR2/1 ligand led to significant inhibition of tumor growth compared to the administration of the control SFV/Luc virus particles. Analysis of the composition of intratumoral lymphoid cells isolated from tumors after SFV/IFNg treatment revealed increased CD4⁺ and CD8⁺ and decreased T-reg (CD4⁺/CD25⁺/FoxP3⁺) cell populations. Furthermore, a significant decrease in the populations of cells bearing myeloid cell markers CD11b, CD38, and CD206 was observed. In conclusion, the SFV/IFNg vector induces a therapeutic antitumor T-cell response and inhibits myeloid cell infiltration in treated tumors. Full article

Alphaviruses are small enveloped viruses with positive-sense RNA genomes. During infection, the alphavirus capsid protein (Cp) selectively packages and assembles with the viral genomic RNA to form the nucleocapsid core, a process critical to the production of infectious virus. Prior studies of the alphavirus Semliki Forest virus (SFV) showed that packaging and assembly are promoted by Cp binding to multiple high affinity sites on the genomic RNA. Here, we developed an in vitro Cp binding assay based on fluorescently labeled RNA oligos. We used this assay to explore the RNA sequence and structure requirements for Cp binding to site #1, the top binding site identified on the genomic RNA during all stages of virus assembly. Our results identify a stem-loop structure that promotes specific binding of the SFV Cp to site #1 RNA. This structure is also recognized by the Cps of the related alphaviruses chikungunya virus and Ross River virus. Full article