Search Results (37)

Flaviviruses are arthropod-borne viruses primarily transmitted through the mosquito Aedes aegypti or Culex genus of mosquitos. These viruses are predominantly found in tropical and subtropical regions of the world with their geographical spread predicted to increase as global temperatures continue to rise. These viruses cause a variety of diseases in humans with the most prevalent being caused by dengue, resulting in hemorrhagic fever and associated sequala. Current approaches for therapeutic control of flavivirus infections are limited, and despite recent advances, there are no approved drugs. Vaccines, available for a few circulating flaviviruses, still have limited potential for controlling contemporary and future outbreaks. Mouse models provide us with a valuable tool to test the effectiveness of drugs and vaccines, yet for many flaviviruses, well-established mouse models are lacking. In this review, we highlight the current state of flavivirus vaccines and therapeutics, as well as our current understanding of mouse models for various flaviviruses. Full article

Dengue virus (DENV) infection, prevalent in tropical and subtropical regions, can progress to dengue hemorrhagic fever (DHF), which increases mortality during secondary infections. DHF is characterized by endothelial damage and vascular leakage. Despite its severity, no specific antiviral treatments exist, and the viral factors responsible for endothelial damage remain unclear. This study examines the role of the DENV envelope protein domain III (EIII) in inducing endothelial apoptosis using a mouse model. Additionally, we aim to explore whether cell death-inducing pathways could serve as drug targets to ameliorate EIII-induced endothelial injury and hemorrhage. In vitro experiments using human endothelial HMEC-1 cells demonstrated that both recombinant EIII (rEIII) and DENV markedly induced caspase-3-mediated endothelial cell death, an effect that was attenuated by co-treatment with chondroitin sulfate B (CSB), N-acetyl cysteine (NAC), and the caspase-3 inhibitor z-DEVD-FMK. In vivo, sequential injections of rEIII and anti-platelet immunoglobulin in mice, designed to mimic the clinical phase of DHF with peak viremia followed by an increase in DENV-induced Ig, including autoantibodies, revealed that these dual treatments markedly triggered caspase-3-dependent apoptosis in vascular endothelial cells at hemorrhage sites. Treatments with z-DEVD-FMK effectively reduced DHF-like symptoms such as thrombocytopenia, hemorrhage, inflammation, hypercoagulation, and endothelial damage. Additionally, CSB and NAC alleviated hemorrhagic symptoms in the mice. These results suggest that targeting EIII, reactive oxygen species, and caspase-3-mediated apoptosis could offer potential therapeutic strategies for addressing EIII-induced hemorrhagic pathogenesis. Full article

The annual number of reported human cases of flavivirus infections continues to increase. Measures taken by local healthcare systems and international organizations are not fully successful. In this regard, new approaches to treatment and prevention of flavivirus infections are relevant. One promising approach is to use monoclonal antibody preparations. The mouse mAb 10H10 is capable of interacting with viruses belonging to the genus Orthoflavivirus which are pathogenic to humans. ELISA and molecular modeling data can indicate that mAb 10H10 recognizes the fusion loop region of E protein. The KD of interaction between the mAb 10H10 and recombinant analogs of the E protein of the tick-borne encephalitis (TBEV), Zika (ZIKV) and dengue (DENV) viruses range from 1.5 to 4 nM. The aim of this study was to map the epitope of this antibody using phage display technology. After three rounds of biopanning, 60 individual phage clones were chosen. The amino acid sequences of the selected peptides were conveniently divided into five groups. Based on the selected peptides, bacteriophages were obtained carrying peptides on the surfaces of the pIII and pVIII proteins, which were tested for binding to the antibody in ELISA. Thus, the epitope of the mAb 10H10 is the highly conserved region 98-DRGWGNXXGLFGK-110 of the flavivirus E protein. The structures of the complexes of the identified peptides with the antibody paratope are proposed using the molecular docking and dynamics methods. Full article

West Nile Virus (WNV) poses a significant global public health threat as a mosquito-borne pathogen. While laboratory mouse models have historically played a crucial role in understanding virus biology, recent research has focused on utilizing immunocompromised models to study arboviruses like dengue and Zika viruses, particularly their interactions with Aedes aegypti mosquitoes. However, there has been a shortage of suitable mouse models for investigating WNV and St. Louis encephalitis virus interactions with their primary vectors, Culex spp. mosquitoes. Here, we establish the AG129 mouse (IFN α/β/γ R^−/−) as an effective vertebrate model for examining mosquito–WNV interactions. Following intraperitoneal injection, AG129 mice exhibited transient viremia lasting several days, peaking on the second or third day post-infection, which is sufficient to infect Culex quinquefasciatus mosquitoes during a blood meal. We also observed WNV replication in the midgut and dissemination to other tissues, including the fat body, in infected mosquitoes. Notably, infectious virions were present in the saliva of a viremic AG129 mouse 16 days post-exposure, indicating successful transmission capacity. These findings highlight the utility of AG129 mice for studying vector competence and WNV–mosquito interactions. Full article

Dengue is a significant public health problem with no specific viral treatment. One of the main challenges in studying dengue is the lack of adequate animal models recapitulating human immune responses. Most studies on humanized mice use NOD-scid IL2R gamma null (NSG) mice, which exhibit poor hematopoiesis for some cell populations. This study compares three humanized (hu) NOD-derived mouse models for dengue virus-2 (DENV-2) infection in the context of human cytokine expression. Three mouse strains (hu-NSG, hu-EXL, and hu-SGM3) received xenotransplants of human CD34+ fetal cord blood cells from a single donor, and one mouse strain received human peripheral blood mononuclear cells (hu-SGM3-PBMCs). All models exhibited infectious viruses in blood confirmed by plaque assay, but mice expressing human cytokines showed higher viremia compared to conventional NSG mice. The hu-SGM3-PBMCs model developed lethal infections, showing a significant increase in viremia and clinical signs. A detectable human cytokine response was observed in all the DENV-2-infected humanized mouse models. In conclusion, humanized NOD-derived mouse models expressing human cytokines offer a relevant platform for the study of dengue pathogenesis and antiviral therapies. Full article

The complete lack of yellow fever virus (YFV) in Asia, and the lack of urban YFV transmission in South America, despite the abundance of the peridomestic mosquito vector Aedes (Stegomyia.) aegypti is an enigma. An immunologically naïve population of over 2 billion resides in Asia, with most regions infested with the urban YF vector. One hypothesis for the lack of Asian YF, and absence of urban YF in the Americas for over 80 years, is that prior immunity to related flaviviruses like dengue (DENV) or Zika virus (ZIKV) modulates YFV infection and transmission dynamics. Here we utilized an interferon α/β receptor knock-out mouse model to determine the role of pre-existing dengue-2 (DENV-2) and Zika virus (ZIKV) immunity in YF virus infection, and to determine mechanisms of cross-protection. We utilized African and Brazilian YF strains and found that DENV-2 and ZIKV immunity significantly suppresses YFV viremia in mice, but may or may not protect relative to disease outcomes. Cross-protection appears to be mediated mainly by humoral immune responses. These studies underscore the importance of re-assessing the risks associated with YF outbreak while accounting for prior immunity from flaviviruses that are endemic. Full article

In search of a mouse model for use in evaluating dengue vaccines, we assessed A129 mice that lacked IFN-α/β receptors, rendering them susceptible to dengue virus (DENV) infection. To our knowledge, no reports have evaluated dengue vaccine efficiency using A129 mice. A129 mice were given a single intraperitoneal (IP) or subcutaneous (SC) injection of the vaccine, Dengvaxia. After 14 days of immunization via the IP or SC injection of Dengvaxia, the A129 mice exhibited notably elevated levels of anti-DENV immunoglobulin G and neutralizing antibodies (NAb) targeting all four DENV serotypes, with DENV-4 displaying the highest NAb levels. After challenge with DENV-2, Dengvaxia and mock-immunized mice survived, while only the mock group exhibited signs of morbidity. Viral genome levels in the serum and tissues (excluding the brain) were considerably lower in the immunized mice compared to those in the mock group. The SC administration of Dengvaxia resulted in lower viremia levels than IP administration did. Therefore, given that A129 mice manifest dengue-related morbidity, including viremia in the serum and other tissues, these mice represent a valuable model for investigating novel dengue vaccines and antiviral drugs and for exploring dengue pathogenesis. Full article

N-(4-hydroxyphenyl) retinamide (4-HPR, or fenretinide) has promising in vitro and in vivo antiviral activity against a range of flaviviruses and an established safety record, but there are challenges to its clinical use. This study evaluated the in vivo exposure profile of a 4-HPR dosage regime previously shown to be effective in a mouse model of severe dengue virus (DENV) infection, comparing it to an existing formulation for human clinical use for other indications and developed/characterised self-emulsifying lipid-based formulations of 4-HPR to enhance 4-HPR in vivo exposure. Pharmacokinetic (PK) analysis comprising single-dose oral and IV plasma concentration-time profiles was performed in mice; equilibrium solubility testing of 4-HPR in a range of lipids, surfactants and cosolvents was used to inform formulation approaches, with lead formulation candidates digested in vitro to analyse solubilisation/precipitation prior to in vivo testing. PK analysis suggested that effective plasma concentrations could be achieved with the clinical formulation, while novel lipid-based formulations achieved > 3-fold improvement. Additionally, 4-HPR exposure was found to be limited by both solubility and first-pass intestinal elimination but could be improved through inhibition of cytochrome P450 (CYP) metabolism. Simulated exposure profiles suggest that a b.i.d dosage regime is likely to maintain 4-HPR above the minimum effective plasma concentration for anti-DENV activity using the clinical formulation, with new formulations/CYP inhibition viable options to increase exposure in the future. Full article

Dengue hemorrhagic fever (DHF) is a severe form of dengue virus (DENV) infection that can lead to abnormal immune responses, endothelial vascular dysfunction, and hemorrhage pathogenesis. The virion-associated envelope protein domain III (EIII) is thought to play a role in the virulence of DENV by damaging endothelial cells. However, it is unclear whether EIII-coated nanoparticles simulating DENV virus particles could cause a more severe pathogenesis than soluble EIII alone. This study aimed to investigate whether EIII-coated silica nanoparticles (EIII-SNPs) could elicit greater cytotoxicity in endothelial cells and hemorrhage pathogenesis in mice compared to EIII or silica nanoparticles alone. The main methods included in vitro assays to assess cytotoxicity and in vivo experiments to examine hemorrhage pathogenesis in mice. EIII-SNPs induced greater endothelial cytotoxicity in vitro than EIII or silica nanoparticles alone. Two-hit combined treatment with EIII-SNPs and antiplatelet antibodies to simulate DHF hemorrhage pathogenesis during secondary DENV infections resulted in higher endothelial cytotoxicity than either treatment alone. In mouse experiments, two-hit combined treatment with EIII-SNPs and antiplatelet antibodies resulted in more severe hemorrhage pathogenesis compared to single treatments of EIII, EIII-SNPs, or antiplatelet antibodies alone. These findings suggest that EIII-coated nanoparticles are more cytotoxic than soluble EIII and could be used to develop a tentative dengue two-hit hemorrhage pathogenesis model in mice. Additionally, our results indicated that EIII-containing DENV particles could potentially exacerbate hemorrhage pathogenesis in DHF patients who have antiplatelet antibodies, highlighting the need for further research on the potential role of EIII in DHF pathogenesis. Full article

To combat infectious diseases, vaccines are considered the best prophylactic strategy for a wide range of the population, but even when vaccines are effective, the administration of therapeutic antibodies against viruses could provide further treatment options, particularly for vulnerable groups whose immunity against the viruses is compromised. Therapeutic antibodies against dengue are ideally engineered to abrogate binding to Fcγ receptors (FcγRs), which can induce antibody-dependent enhancement (ADE). However, the Fc effector functions of neutralizing antibodies against SARS-CoV-2 have recently been reported to improve post-exposure therapy, while they are dispensable when administered as prophylaxis. Hence, in this report, we investigated the influence of Fc engineering on anti-virus efficacy using the anti-dengue/Zika human antibody SIgN-3C and found it affected the viremia clearance efficacy against dengue in a mouse model. Furthermore, we demonstrated that complement activation through antibody binding to C1q could play a role in anti-dengue efficacy. We also generated a novel Fc variant, which displayed the ability for complement activation but showed very low FcγR binding and an undetectable level of the risk of ADE in a cell-based assay. This Fc engineering approach could make effective and safe anti-virus antibodies against dengue, Zika and other viruses. Full article

Antibody-dependent enhancement of infection (ADE) is clinically relevant to Dengue virus (DENV) infection and poses a major risk to the application of monoclonal antibody (mAb)-based therapeutics against related flaviviruses such as the Zika virus (ZIKV). Here, we tested a two-tier approach for selecting non-cross-reactive mAbs combined with modulating Fc glycosylation as a strategy to doubly secure the elimination of ADE while preserving Fc effector functions. To this end, we selected a ZIKV-specific mAb (ZV54) and generated three ZV54 variants using Chinese hamster ovary cells and wild-type (WT) and glycoengineered ΔXF Nicotiana benthamiana plants as production hosts (ZV54^CHO, ZV54^WT, and ZV54^ΔXF). The three ZV54 variants shared an identical polypeptide backbone, but each exhibited a distinct Fc N-glycosylation profile. All three ZV54 variants showed similar neutralization potency against ZIKV but no ADE activity for DENV infection, validating the importance of selecting the virus/serotype-specific mAbs for avoiding ADE by related flaviviruses. For ZIKV infection, however, ZV54^CHO and ZV54^ΔXF showed significant ADE activity while ZV54^WT completely forwent ADE, suggesting that Fc glycan modulation may yield mAb glycoforms that abrogate ADE even for homologous viruses. In contrast to the current strategies for Fc mutations that abrogate all effector functions along with ADE, our approach allowed the preservation of effector functions as all ZV54 glycovariants retained antibody-dependent cellular cytotoxicity (ADCC) against the ZIKV-infected cells. Furthermore, the ADE-free ZV54^WT demonstrated in vivo efficacy in a ZIKV-infection mouse model. Collectively, our study provides further support for the hypothesis that antibody–viral surface antigen and Fc-mediated host cell interactions are both prerequisites for ADE, and that a dual-approach strategy, as shown herein, contributes to the development of highly safe and efficacious anti-ZIKV mAb therapeutics. Our findings may be impactful to other ADE-prone viruses, including SARS-CoV-2. Full article

Dengue virus serotypes 1 to 4 (DENV1–4) place nearly half the global population at risk of infection and the licenced tetravalent dengue vaccine fails to protect individuals who have not previously been exposed to DENV. The development of intervention strategies had long been hampered by the lack of a suitable small animal model. DENV does not replicate in wild-type mice due to its inability to antagonise the mouse type I interferon (IFN) response. Mice deficient in type I IFN signalling (Ifnar1^−/− mice) are highly susceptible to DENV infection, but their immunocompromised status makes it difficult to interpret immune responses elicited by experimental vaccines. To develop an alternative mouse model for vaccine testing, we treated adult wild-type mice with MAR1-5A3—an IFNAR1-blocking, non-cell-depleting antibody—prior to infection with the DENV2 strain D2Y98P. This approach would allow for vaccination of immunocompetent mice and subsequent inhibition of type I IFN signalling prior to challenge infection. While Ifnar1^−/− mice quickly succumbed to infection, MAR1-5A3-treated mice did not show any signs of illness but eventually seroconverted. Infectious virus was recovered from the sera and visceral organs of Ifnar1^−/− mice, but not from those of mice treated with MAR1-5A3. However, high levels of viral RNA were detected in the samples of MAR1-5A3-treated mice, indicating productive viral replication and dissemination. This transiently immunocompromised mouse model of DENV2 infection will aid the pre-clinical assessment of next-generation vaccines as well as novel antiviral treatments. Full article

The four serotypes of dengue virus (DENV1–4) continue to pose a major public health threat. The first licenced dengue vaccine, which expresses the surface proteins of DENV1–4, has performed poorly in immunologically naïve individuals, sensitising them to antibody-enhanced dengue disease. DENV non-structural protein 1 (NS1) can directly induce vascular leakage, the hallmark of severe dengue disease, which is blocked by NS1-specific antibodies, making it an attractive target for vaccine development. However, the intrinsic ability of NS1 to trigger vascular leakage is a potential drawback of its use as a vaccine antigen. Here, we modified DENV2 NS1 by mutating an N-linked glycosylation site associated with NS1-induced endothelial hyperpermeability and used modified vaccinia virus Ankara (MVA) as a vector for its delivery. The resulting construct, rMVA-D2-NS1-N207Q, displayed high genetic stability and drove efficient secretion of NS1-N207Q from infected cells. Secreted NS1-N207Q was composed of dimers and lacked N-linked glycosylation at position 207. Prime–boost immunisation of C57BL/6J mice induced high levels of NS1-specific antibodies binding various conformations of NS1 and elicited NS1-specific CD4⁺ T-cell responses. Our findings support rMVA-D2-NS1-N207Q as a promising and potentially safer alternative to existing NS1-based vaccine candidates, warranting further pre-clinical testing in a relevant mouse model of DENV infection. Full article

Arboviruses (an acronym for “arthropod-borne virus”), such as dengue, yellow fever, Zika, and Chikungunya, are important human pathogens transmitted by mosquitoes. These viruses impose a growing burden on public health. Despite laboratory mice having been used for decades for understanding the basic biological phenomena of these viruses, it was only recently that researchers started to develop immunocompromised animals to study the pathogenesis of arboviruses and their transmission in a way that parallels natural cycles. Here, we show that the AG129 mouse (IFN α/β/γ R^−/−) is a suitable and comprehensive vertebrate model for studying the mosquito vector competence for the major arboviruses of medical importance, namely the dengue virus (DENV), yellow fever virus (YFV), Zika virus (ZIKV), Mayaro virus (MAYV), and Chikungunya virus (CHIKV). We found that, after intraperitoneal injection, AG129 mice developed a transient viremia lasting several days, peaking on day two or three post infection, for all five arboviruses tested in this study. Furthermore, we found that the observed viremia was ample enough to infect Aedes aegypti during a blood meal from the AG129 infected mice. Finally, we demonstrated that infected mosquitoes could transmit each of the tested arboviruses back to naïve AG129 mice, completing a full transmission cycle of these vector-borne viruses. Together, our data show that A129 mice are a simple and comprehensive vertebrate model for studies of vector competence, as well as investigations into other aspects of mosquito biology that can affect virus–host interactions. Full article

Since the first isolation in 1943, the dengue virus (DENV) has spread throughout the world, but effective antiviral drugs or vaccines are still not available. To provide a more stable reporter DENV for vaccine development and antiviral drug screening, we constructed a reporter DENV containing the NanoLuc reporter gene, which was inserted into the 5′ untranslated region and capsid junction region, enabling rapid virus rescue by in vitro ligation. In addition, we established a live imaging mouse model and found that the reporter virus maintained the neurovirulence of prototype DENV before engineering. DENV-4 exhibited dramatically increased neurovirulence following a glycosylation site-defective mutation in the envelope protein. Significant mice mortality with neurological onset symptoms was observed after intracranial infection of wild-type (WT) mice, thus providing a visualization tool for DENV virulence assessment. Using this model, DENV was detected in the intestinal tissues of WT mice after infection, suggesting that intestinal lymphoid tissues play an essential role in DENV pathogenesis. Full article