AG129 Mice Research Articles

7D, a small molecule inhibits dengue infection by increasing interferons and neutralizing-antibodies via CXCL4:CXCR3:p38:IRF3 and Sirt1:STAT3 axes respectively

There are a limited number of effective vaccines against dengue virus (DENV) and significant efforts are being made to develop potent anti-virals. Previously, we described that platelet-chemokine CXCL4 negatively regulates interferon (IFN)-α/β synthesis and promotes DENV2 replication. An antagonist to CXCR3 (CXCL4 receptor) reversed it and inhibited viral replication. In a concurrent search, we identified CXCR3-antagonist from our compound library, namely 7D, which inhibited all serotypes of DENV in vitro. With a half-life of ~2.85 h in plasma and no significant toxicity, 7D supplementation (8 mg/kg-body-weight) to DENV2-infected IFNα/β/γR−/−AG129 or wild-type C57BL6 mice increased synthesis of IFN-α/β and IFN-λ, and rescued disease symptoms like thrombocytopenia, leukopenia and vascular-leakage, with improved survival. 7D, having the property to inhibit Sirt-1 deacetylase, promoted acetylation and phosphorylation of STAT3, which in-turn increased plasmablast proliferation, germinal-center maturation and synthesis of neutralizing-antibodies against DENV2 in mice. A STAT3-inhibitor successfully inhibited these effects of 7D. Together, these observations identify compound 7D as a stimulator of IFN-α/β/λ synthesis via CXCL4:CXCR3:p38:IRF3 signaling, and a booster for neutralizing-antibody generation by promoting STAT3-acetylation in plasmablasts, capable of protecting dengue infection.

A robust mouse model of HPIV-3 infection and efficacy of GS-441524 against virus-induced lung pathology

Human parainfluenza virus type 3 (HPIV-3) can cause severe respiratory tract infections. There are no convenient small-animal infection models. Here, we show viral replication in the upper and lower airways of AG129 mice (double IFNα/β and IFNγ receptor knockout mice) upon intranasal inoculation. By multiplex fluorescence RNAscope and immunohistochemistry followed by confocal microscopy, we demonstrate viral tropism to ciliated cells and club cells of the bronchiolar epithelium. HPIV-3 causes a marked lung pathology. No virus transmission of the virus was observed by cohousing HPIV-3-infected AG129 mice with other mice. Oral treatment with GS-441524, the parent nucleoside of remdesivir, reduced infectious virus titers in the lung, with a relatively normal histology. Intranasal treatment also affords an antiviral effect. Thus, AG129 mice serve as a robust preclinical model for developing therapeutic and prophylactic strategies against HPIV-3. We suggest further investigation of GS-441524 and its prodrug forms to treat HPIV-3 infection in humans.

Suitable Mouse Model to Study Dynamics of West Nile Virus Infection in Culex quinquefasciatus Mosquitoes.

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.

Characterization of Effect of Enterovirus D68 in 129/Sv Mice Deficient in IFN-α/β and/or IFN-γ Receptors.

Enterovirus D68 (EV-D68), a respiratory RNA virus in the family Picornaviridae, is implicated as a potential etiological agent for acute flaccid myelitis in preteen adolescents. The absence of a specific therapeutic intervention necessitates the development of an effective animal model for EV-D68. The AG129 mouse strain, characterized by the double knockout of IFN-α/β and IFN-γ receptors on the 129 genetic background, has been proposed as a suitable model for EV-D68. The goals of this study were to assess the effect of a nonmouse-adapted EV-D68 strain (US/MO/14-18947, NR-49129) in AG129 (IFN-α/β and IFN-γ receptors null), A129 (IFN-α/β receptor null), G129 (IFN-γ receptor null), and the 129 background strain (129S2/SvPasCrl) when infected intraperitoneally at 10 d of age. Both AG129 and A129 strains demonstrated similar clinical signs (paralysis, paresis, lethargy, dyspnea [characterized by prominent abdominal respiration], and morbidity requiring euthanasia) induced by EV-D68. While G129 and 129S2 strains also exhibited susceptibility to EV-D68, the severity of clinical signs was less than in AG129 and A129 strains, and many survived to the experimental endpoint. Histopathological and immunohistochemical data confirmed EV-D68 tropism for the skeletal muscle and spinal cord and suggest that the dyspnea observed in infected mice could be attributed, in part, to lesions in the diaphragmatic skeletal muscles. These findings contribute valuable insights into the pathogenesis of EV-D68 infection in this mouse model and provide investigators with key information on virus dose and mouse strain selection when using this mouse model to evaluate candidate EV-D68 therapeutics.

The inoculum dose of Zika virus can affect the viral replication dynamics, cytokine responses and survival rate in immunocompromised AG129 mice

Zika virus, a mosquito-borne arbovirus, has repeatedly caused large pandemics with symptoms worsening from mild and self-limiting diseases to Guillain–Barré syndrome in adults and fetal microcephaly in newborns. In recent years, Zika virus diseases have posed a serious threat to human health. The shortage of susceptible small animal models makes it difficult to study pathogenic mechanisms and evaluate potential therapies for Zika virus infection. Therefore, we chose immunocompromised mice (AG129 mice) deficient in IFN-α/β and IFN-γ receptors, which can abolish the innate immune system that prevents Zika virus infection early. AG129 mice were infected with the Zika virus, and this mouse model exhibited replication dynamics, tissue tropism, pathological lesion and immune activation of the Zika virus. Our results suggest that the inoculum dose of Zika virus can affect the viral replication dynamics, cytokine responses and survival rate in AG129 mice. By testing the potential antiviral drug favipiravir, several critical indicators, including replication dynamics and survival rates, were identified in AG129 mice after Zika virus infection. It is suggested that the model is reliable for drug evaluation. In brief, this model provides a potential platform for studies of the infectivity, virulence, and pathogenesis of the Zika virus. Moreover, the development of an accessible mouse model of Zika virus infection will expedite the research and deployment of therapeutics and vaccines.

Parallel Multifactorial Process Optimization and Intensification for High-Yield Production of Live YF17D-Vectored Zika Vaccine.

The live-attenuated yellow fever 17D strain is a potent vaccine and viral vector. Its manufacture is based on embryonated chicken eggs or adherent Vero cells. Both processes are unsuitable for rapid and scalable supply. Here, we introduce a high-throughput workflow to identify suspension cells that are fit for the high-yield production of live YF17D-based vaccines in an intensified upstream process. The use of an automated parallel ambr15 microbioreactor system for screening and process optimization has led to the identification of two promising cell lines (AGE1.CR.pIX and HEKDyn) and the establishment of optimized production conditions, which have resulted in a >100-fold increase in virus titers compared to the current state of the art using adherent Vero cells. The process can readily be scaled up from the microbioreactor scale (15 mL) to 1 L stirred tank bioreactors. The viruses produced are genetically stable and maintain their favorable safety and immunogenicity profile, as demonstrated by the absence of neurovirulence in suckling BALB/c mice and consistent seroprotection in AG129 mice. In conclusion, the presented workflow allows for the rapid establishment of a robust, scalable, and high-yield process for the production of live-attenuated orthoflavivirus vaccines, which outperforms current standards. The approach described here can serve as a model for the development of scalable processes and the optimization of yields for other virus-based vaccines that face challenges in meeting growing demands.

Tanreqing injection demonstrates anti-dengue activity through the regulation of the NF-κB-ICAM-1/VCAM-1 axis

BackgroundTanreqing injection (TRQ) has been employed in clinical practice as a treatment for dengue fever (DF). Nevertheless, the precise pharmacological mechanism underlying its efficacy remains elusive. MethodNetwork pharmacology, molecular docking, transcriptome sequencing, and experimental evaluation were employed to analyze and study the inhibitory potential of TRQ against dengue virus (DENV). ResultWe found that TRQ inhibited the replication of DENV in human umbilical vein endothelial cells, Huh-7 cells, and Hep3B cells. In addition, TRQ prolonged the survival duration of AG129 mice infected with DF, decreased the viral load in serum and organs, and alleviated organ damage. Subsequently, ultra-high-performance liquid chromatography-tandem mass spectrometry analysis of TRQ was performed to identify 314 targets associated with 36 active compounds present in TRQ. Integration of multiple databases yielded 47 DF-related genes. Then, 15 hub targets of TRQ in DF were determined by calculating the network topology parameters (Degree). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that these pathways were primarily enriched in the processes of cytokine activation and leukocyte cross-endothelial migration, with significant enrichment of cell adhesion molecules. Molecular docking revealed favorable binding affinity between TRQ's key active compounds and the predicted hub targets. Transcriptome sequencing results showed TRQ's ability to restore the expression of vascular cell adhesion molecule-1 (VCAM-1) post-DENV infection. Finally, TRQ was found to modulate the immune status by regulating the nuclear factor kappa-B (NF-κB)- intercellular cell adhesion molecule-1 (ICAM-1)/VCAM-1 axis, as well as reduce immune cell alterations, inflammatory factor secretion, vascular permeability, and bleeding tendencies induced by DENV infection. ConclusionOur research suggests that TRQ exerts therapeutic effects on DF by regulating the NF-κB-ICAM-1/VCAM-1 axis.

The High Capacity of Brazilian Aedes aegypti Populations to Transmit a Locally Circulating Lineage of Chikungunya Virus.

The incidence of chikungunya has dramatically surged worldwide in recent decades, imposing an expanding burden on public health. In recent years, South America, particularly Brazil, has experienced outbreaks that have ravaged populations following the rapid dissemination of the chikungunya virus (CHIKV), which was first detected in 2014. The primary vector for CHIKV transmission is the urban mosquito species Aedes aegypti, which is highly prevalent throughout Brazil. However, the impact of the locally circulating CHIKV genotypes and specific combinations of local mosquito populations on vector competence remains unexplored. Here, we experimentally analyzed and compared the infectivity and transmissibility of the CHIKV-ECSA lineage recently isolated in Brazil among four Ae. aegypti populations collected from different regions of the country. When exposed to CHIKV-infected AG129 mice for blood feeding, all the mosquito populations displayed high infection rates and dissemination efficiency. Furthermore, we observed that all the populations were highly efficient in transmitting CHIKV to a vertebrate host (naïve AG129 mice) as early as eight days post-infection. These results demonstrate the high capacity of Brazilian Ae. aegypti populations to transmit the locally circulating CHIKV-ECSA lineage. This observation could help to explain the high prevalence of the CHIKV-ECSA lineage over the Asian lineage, which was also detected in Brazil in 2014. However, further studies comparing both lineages are necessary to gain a better understanding of the vector's importance in the epidemiology of CHIKV in the Americas.

Establishment of a lethal mouse model of emerging tick-borne orthonairovirus infections.

Emerging and reemerging tick-borne virus infections caused by orthonairoviruses (family Nairoviridae), which are genetically distinct from Crimean-Congo hemorrhagic fever virus, have been recently reported in East Asia. Here, we have established a mouse infection model using type-I/II interferon receptor-knockout mice (AG129 mice) both for a better understanding of the pathogenesis of these infections and validation of antiviral agents using Yezo virus (YEZV), a novel orthonairovirus causing febrile illnesses associated with tick bites in Japan and China. YEZV-inoculated AG129 mice developed hepatitis with body weight loss and died by 6 days post infection. Blood biochemistry tests showed elevated liver enzyme levels, similar to YEZV-infected human patients. AG129 mice treated with favipiravir survived lethal YEZV infection, demonstrating the anti-YEZV effect of this drug. The present mouse model will help us better understand the pathogenicity of the emerging tick-borne orthonairoviruses and the development of specific antiviral agents for their treatment.

Fingolimod (FTY720), an FDA-approved sphingosine 1-phosphate (S1P) receptor agonist, restores endothelial hyperpermeability in cellular and animal models of dengue virus serotype 2 infection.

Extensive vascular leakage and shock is a major cause of dengue-associated mortality. At present, there are no specific treatments available. Sphingolipid pathway is a key player in the endothelial barrier integrity; and is mediated through the five sphingosine-1-phosphate receptors (S1PR1-S1PR5). Signaling through S1PR2 promotes barrier disruption; and in Dengue virus (DENV)-infection, there is overexpression of this receptor. Fingolimod (FTY720) is a specific agonist that targets the remaining barrier-protective S1P receptors, without targeting S1PR2. In the present study, we explored whether FTY720 treatment can alleviate DENV-induced endothelial hyperpermeability. In functional assays, in both in vitro systems and in AG129 animal models, FTY720 treatment was found effective. Upon treatment, there was complete restoration of the monolayer integrity in DENV serotype 2-infected human microvascular endothelial cells (HMEC-1). At the molecular level, the treatment reversed activation of the S1P pathway. It significantly reduced the phosphorylation of the key molecules such as PTEN, RhoA, and VE-Cadherin; and also, the expression levels of S1PR2. In DENV2-infected AG129 mice treated with FTY720, there was significant improvement in weight gain, in overall clinical symptoms, and in survival. Whereas 100% of the DENV2-infected, untreated animals died by day-10 post-infection, 70% of the FTY720-treated animals were alive; and at the end of the 15-day post-infection observation period, 30% of them were still surviving. There was a significant reduction in the Evan's-blue dye permeability in the organs of FTY720-treated, DENV-2 infected animals; and also improvement in the hemogram, with complete restoration of thrombocytopenia and hepatic function. Our results show that the FDA-approved molecule Fingolimod (FTY720) is a promising therapeutic intervention in severe dengue.

Dengue virus infection in mice induces bone marrow myeloid cell differentiation and generates Ly6Glow immature neutrophils with modulated functions.

While neutrophil activation during dengue virus infection is known, the effect of dengue virus infection on neutrophil biogenesis has not been studied. We demonstrate that dengue virus serotype 2 induces the differentiation of mice progenitor cells ex vivo toward the CD11b+Ly6C+Ly6G+ granulocyte population. We further observed an expansion of CD11b+Ly6CintLy6Glow myeloid cells in the bone marrow of dengue virus serotype 2-infected AG129 mice with low CXCR2 expression, implying an immature population. Additionally, dengue virus serotype 2 alone could induce the differentiation of promyelocyte cell line HL-60 into neutrophil-like cells, as evidenced by increased expression of CD10, CD66b, CD16, CD11b, and CD62L, corroborating the preferential shift toward neutrophil differentiation by dengue virus serotype 2 in the mouse model of dengue infection. The functional analysis showed that dengue virus serotype 2-induced neutrophil-like cells exhibited reduced phagocytic activity and enhanced NETosis, as evidenced by the increased production of myeloperoxidase, citrullinated histones, extracellular DNA, and superoxide. These neutrophil-like cells lose their ability to proliferate irreversibly and undergo arrest in the G0 to G1 phase of the cell cycle. Further studies show that myeloperoxidase-mediated signaling operating through the reactive oxygen species axis may be involved in dengue virus serotype 2-induced proliferation and differentiation of bone marrow cells as ABAH, a myeloperoxidase inhibitor, limits cell proliferation in vitro and ex vivo, affects the cell cycle, and reduces reactive oxygen species production. Additionally, myeloperoxidase inhibitor reduced NETosis and vascular leakage in dengue virus serotype 2-infected AG129 mice. Our study thus provides evidence that dengue virus serotype 2 can accelerate the differentiation of bone marrow progenitor cells into neutrophils through myeloperoxidase and modulate their functions.

Liang-Ge-San inhibits dengue virus serotype 2 infection by reducing caveolin1-induced cytoplasmic heat shock protein 70 translocation into the plasma membrane

Dengue virus (DENV) is a major public health threat. However, there are no specific therapeutic drugs for DENV. Many Chinese heat-cleaning formulas, such as Liang-Ge-San (LGS), have been frequently used in the virus-induced diseases. The antiviral effect of LGS has not been reported yet. In this study, the effect of LGS on the inhibition of dengue virus serotype 2 (DENV-2) was investigated and the relevant mechanism was explored. High-performance liquid chromatography was applied to analyze the chemical characterization of LGS. The in vitro antiviral activities of LGS against DENV-2 were evaluated by time-of-drug-addition assay. The binding of heat shock protein 70 (Hsp70) and envelope (E) protein or caveolin1 (Cav1) were analyzed by immunofluorescence and immunoprecipitation assays. Then the role of Cav1 in the anti-DENV-2 effects of LGS was further examined. DENV-2 infected Institute of Cancer Research suckling mice (n=10) and AG129 mice (n=8) were used to examine the protective effects of LGS. It was found that geniposide, liquiritin, forsythenside A, forsythin, baicalin, baicalein, rhein, and emodin maybe the characteristic components of LGS. LGS inhibited the early stage of DENV-2 infection, decreased the expression levels of viral E and non-structural protein 1 (NS1) proteins. LGS also reduced E protein and Hsp70 binding and attenuated the translocation of Hsp70 from cytoplasm to the cell membrane. Moreover, LGS decreased the binding of Hsp70 to Cav1. Further study showed that the overexpression of Cav1 reversed LGS-mediated E protein and Hsp70 inhibition in the plasma membrane. In the in vivo study, LGS was highly effective in prolonging the survival time, reducing viral loads. This work demonstrates for the first time that LGS exerts anti-DENV-2 activity in vitro and in vivo. LGS decreases DENV-2-stimulated cytoplasmic Hsp70 translocation into the plasma membrane by Cav1 inhibition, thereby inhibiting the early stage of virus infection. These findings indicate that LGS may be a candidate for the treatment of DENV.

The Role of Interferon Receptors α/β/γ Ablation During Western Diet-Induced Obesity and Insulin Resistance in the Inflectional Model AG129 Mice Strain.

Diet-induced obesity triggers elevation of circulating pro-inflammatory cytokines and acute-phase proteins, including interferons (IFNs). IFNs strongly contribute to low-grade inflammation associated with obesity-related complications, such as nonalcoholic fat liver disease and diabetes. In this study, AG129 mice model (double-knockout strain for IFN α/β/γ receptors) was fed with a high-fat high-sucrose (HFHS) diet (Western diet) for 20 weeks aiming to understand the impact of IFN receptor ablation on diet-induced obesity, insulin resistance, and nonalcoholic fat liver disease. Mice were responsive to the diet, becoming obese after 20 weeks of HFHS diet which was accompanied by 2-fold increase of white adipose tissues. Moreover, animals developed glucose and insulin intolerance, as well as dysregulation of insulin signaling mediators such as Insulin Receptor Substrate 1 (IRS1), protein kinase B (AKT), and S6 ribosomal protein. Liver increased interstitial cells, and lipid accumulation was also found, presenting augmented fibrotic markers (transforming growth factor beta 1 [Tgfb1], Keratin 18 [Krt18], Vimentin [Vim]), yet lower expression on IFN receptor downstream proteins (Toll-like receptor [TLR] 4, nuclear factor kappa-light-chain-enhancer of activated B cells [NFκB], and cAMP response element-binding protein [CREB]). Thus, IFN receptor ablation promoted effects on NFκB and CREB pathways, with no positive effects on systemic homeostasis in diet-induced obese mice. Therefore, we conclude that IFN receptor signaling is not essential for promoting the complications of diet-induced obesity and thus cannot be correlated with metabolic diseases in a noninfectious condition.

Higher-temperature-adapted dengue virus serotype 2 strain exhibits enhanced virulence in AG129 mouse model.

The factors that drive dengue virus (DENV) evolution, and selection of virulent variants are yet not clear. Higher environmental temperature shortens DENV extrinsic incubation period in mosquitoes, increases human transmission, and plays a critical role in outbreak dynamics. In the present study, we looked at the effect of temperature in altering the virus virulence. We found that DENV cultured at a higher temperature in C6/36 mosquito cells was significantly more virulent than the virus grown at a lower temperature. In a mouse model, the virulent strain induced enhanced viremia and aggressive disease with a short course, hemorrhage, severe vascular permeability, and death. Higher inflammatory cytokine response, thrombocytopenia, and severe histopathological changes in vital organs such as heart, liver, and kidney were hallmarks of the disease. Importantly, it required only a few passages for the virus to acquire a quasi-species population harboring virulence-imparting mutations. Whole genome comparison with a lower temperature passaged strain identified key genomic changes in the structural protein-coding regions as well as in the 3'UTR of the viral genome. Our results point out that virulence-enhancing genetic changes could occur in the dengue virus genome under enhanced growth temperature conditions in mosquito cells.

Cholesterol-Lowering Drugs as Potential Antivirals: A Repurposing Approach against Flavivirus Infections.

Flaviviruses, including Dengue (DENV), Zika (ZIKV), and Yellow Fever (YFV) viruses, represent a significant global health burden. The development of effective antiviral therapies against these viruses is crucial to mitigate their impact. This study investigated the antiviral potential of the cholesterol-lowering drugs atorvastatin and ezetimibe in monotherapy and combination against DENV, ZIKV, and YFV. In vitro results demonstrated a dose-dependent reduction in the percentage of infected cells for both drugs. The combination of atorvastatin and ezetimibe showed a synergistic effect against DENV 2, an additive effect against DENV 4 and ZIKV, and an antagonistic effect against YFV. In AG129 mice infected with DENV 2, monotherapy with atorvastatin or ezetimibe significantly reduced clinical signs and increased survival. However, the combination of both drugs did not significantly affect survival. This study provides valuable insights into the potential of atorvastatin and ezetimibe as antiviral agents against flaviviruses and highlights the need for further investigations into their combined therapeutic effects.

Neutralizing antibodies targeting a novel epitope on envelope protein exhibited broad protection against flavivirus without risk of disease enhancement

BackgroundFlavivirus causes many serious public health problems worldwide. However, licensed DENV vaccine has restrictions on its use, and there is currently no approved ZIKV vaccine. Development of a potent and safe flavivirus vaccine is urgently needed. As a previous study revealed the epitope, RCPTQGE, located on the bc loop in the E protein domain II of DENV, in this study, we rationally designed and synthesized a series of peptides based on the sequence of JEV epitope RCPTTGE and DENV/ZIKV epitope RCPTQGE.MethodsImmune sera were generated by immunization with the peptides which were synthesized by using five copies of RCPTTGE or RCPTQGE and named as JEV-NTE and DV/ZV-NTE. Immunogenicity and neutralizing abilities of JEV-NTE or DV/ZV-NTE-immune sera against flavivirus were evaluated by ELISA and neutralization tests, respectively. Protective efficacy in vivo were determined by passive transfer the immune sera into JEV-infected ICR or DENV- and ZIKV-challenged AG129 mice. In vitro and in vivo ADE assays were used to examine whether JEV-NTE or DV/ZV-NTE-immune sera would induce ADE.ResultsPassive immunization with JEV-NTE-immunized sera or DV/ZV-NTE-immunized sera could increase the survival rate or prolong the survival time in JEV-challenged ICR mice and reduce the viremia levels significantly in DENV- or ZIKV-infected AG129 mice. Furthermore, neither JEV -NTE- nor DV/ZV-NTE-immune sera induced antibody-dependent enhancement (ADE) as compared with the control mAb 4G2 both in vitro and in vivo.ConclusionsWe showed for the first time that novel bc loop epitope RCPTQGE located on the amino acids 73 to 79 of DENV/ZIKV E protein could elicit cross-neutralizing antibodies and reduced the viremia level in DENV- and ZIKV-challenged AG129 mice. Our results highlighted that the bc loop epitope could be a promising target for flavivirus vaccine development.

Identification of an effective fraction from Ampelopsis Radix with anti-dengue virus activities in vitro and in vivo.

Dengue virus (DENV) infection is a global public health issue without effective therapeutic interventions. Chinese medicine with heat-clearing and detoxifying properties has been frequently used in the treatment of viral infection. Ampelopsis Radix (AR) is a traditional Chinese medicine for clearing heat and detoxification that has been widely used in the prevention and treatment of infectious diseases. However, no studies on the effects of AR against viral infection have been reported, thus far. To explore the anti-DENV activities of the fraction (AR-1) obtained from AR both in vitro and in vivo. The chemical composition of AR-1 was identified by liquid chromatography-tandem MS (LC‒MS/MS). The antiviral activities of AR-1 were studied in baby hamster kidney fibroblast BHK-21cells, ICR suckling mice and induction of interferon α/β (IFN-α/β) and IFN-γ R-/- (AG129) mice. Based on LC‒MS/MS analysis, 60 compounds (including flavonoids, phenols, anthraquinones, alkaloids and other types) were tentatively characterized from AR-1. AR-1 inhibited the cytopathic effect, the production of progeny virus and the synthesis of viral RNA and proteins by blocking DENV-2 binding to BHK-21cells. Moreover, AR-1 significantly attenuated weight loss, decreased clinical scores and prolonged the survival of DENV-infected ICR suckling mice. Critically, the viral load in blood, brain and kidney tissues and the pathological changes in brain were remarkably alleviated after AR-1 treatment. Further study on AG129 mice showed that AR-1 obviously improved the clinical manifestations and survival rate, reduced viremia, attenuated gastric distension and relieved the pathological lesions caused by DENV. In summary, this is the first report that AR-1 exhibits anti-DENV effects both in vitro and in vivo, which suggests that AR-1 may be developed as a therapeutic candidate against DENV infection.

Diagnostic and vaccine potential of Zika virus envelope protein (E) derivates produced in bacterial and insect cells.

In the present study we evaluated the features of different recombinant forms of Zika virus (ZIKV) proteins produced in either bacterial (Eschericha coli) or insect cells (Drosophila melanogaster). The ZIKV-envelope glycoprotein (EZIKV) is responsible for virus entry into host cells, is the main target of neutralizing antibodies and has been used as a target antigen either for serological tests or for the development of subunit vaccines. The EZIKV is composed of three structural and functional domains (EDI, EDII, and EDIII), which share extensive sequence conservation with the corresponding counterparts expressed by other flaviviruses, particularly the different dengue virus (DENV) subtypes. In this study, we carried out a systematic comparison of the antigenicity and immunogenicity of recombinant EZIKV, EDI/IIZIKV and EDIIIZIKV produced in E. coli BL21 and Drosophila S2 cells. For the antigenicity analysis we collected 88 serum samples from ZIKV-infected participants and 57 serum samples from DENV-infected. For immunogenicity, C57BL/6 mice were immunized with two doses of EZIKV, EDI/IIZIKV and EDIIIZIKV produced in E. coli BL21 and Drosophila S2 cells to evaluate humoral and cellular immune response. In addition, AG129 mice were immunized with EZIKV and then challenge with ZIKV. Testing of samples collected from ZIKV-infected and DENV-infected participants demonstrated that the EZIKV and EDIIIZIKV produced in BL21 cells presented better sensitivity and specificity compared to proteins produced in S2 cells. In vivo analyses were carried out with C57BL/6 mice and the results indicated that, despite similar immunogenicity, antigens produced in S2 cells, particularly EZIKV and EDIIIZIKV, induced higher ZIKV-neutralizing antibody levels in vaccinated mice. In addition, immunization with EZIKV expressed in S2 cells delayed the onset of symptoms and increased survival rates in immunocompromised mice. All recombinant antigens, either produced in bacteria or insect cells, induced antigen-specific CD4+ and CD8+ T cell responses. In conclusion, the present study highlights the differences in antigenicity and immunogenicity of recombinant ZIKV antigens produced in two heterologous protein expression systems.

Human IVIG treatment in a neurological disease model for Enterovirus A71 infection in 28-day-old AG129 mice

Enterovirus A71 can cause serious neurological disease in young children. Animal models for EV-A71 are needed to evaluate potential antiviral therapies. Existing models have limitations, including lack of lethality or crucial disease signs. Here we report the development of an EV-A71 model in 28-day-old mice. Virus was serially passaged until it produced consistent lethality and rear-limb paralysis. Onset of disease occurred between days 6–9 post-infection, with mortality following weight loss and neurological signs on days 9–14. In addition, a single administration of human intravenous immunoglobulin at doses of 200, 400 and 800 mg/kg at 4h post-infection was evaluated in the model. Protection from weight loss, neurological signs, and mortality (between 50 and 89%) were observed at doses of 400 mg/kg or greater. Based on these results, IVIG was selected for use as a positive control in this acute model, and suggest that IVIG is a potential therapeutic for EV-A71 infections.

Sinococuline, a bioactive compound of Cocculus hirsutus has potent anti-dengue activity

Dengue virus (DENV) infection has increased worldwide, with over 400 million infections annually, and has become a serious public health concern. Several drug candidates, new and repurposed, have failed to meet the primary efficacy endpoints. We have recently shown that Aqueous Extract of the stem of Cocculus hirsutus (AQCH) was effective in vitro and in vivo against DENV and was safe in humans. We now report that an active ingredient of AQCH, Sinococuline, protects against the antibody-mediated secondary-DENV infection in the AG129 mouse model. DENV infection markers were assessed, viz. serum viremia and vital organs pathologies-viral load, proinflammatory cytokines and intestinal vascular leakage. The treatment with Sinococuline at 2.0 mg/kg/day; BID (twice a day), was the most effective in protecting the severely DENV-infected AG129 mice. Also, this dose effectively reduced serum viremia and tissue-viral load and inhibited the elevated expression levels of proinflammatory cytokines (TNF-α and IL-6) in several vital organs. Based on these findings, it could be explored further for pre-clinical and clinical developments for the treatment of dengue.