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As emerging and re-emerging pathogens, filoviruses, especially Ebola virus (EBOV), pose a great threat to public health and require sustained attention and ongoing surveillance. More vaccines and antiviral drugs are imperative to be developed and stockpiled to respond to unpredictable outbreaks. Virus-like vesicles, generated by alphavirus replicons expressing homogeneous or heterogeneous glycoproteins, have demonstrated the capacity of self-propagation and shown great potential in vaccine development. Here, we describe a novel class of Ebola virus-like vesicles (eVLVs) incorporating both EBOV GP and VP40. The eVLVs exhibited similar antigenicity as EBOV. In murine models, eVLVs were highly attenuated and elicited robust GP-specific antibodies with neutralizing activities. Importantly, a single dose of eVLVs conferred complete protection in a surrogate EBOV lethal mouse model. Furthermore, our VLVs strategy was also successfully applied to Marburg virus (MARV), the representative member of the genus Marburgvirus. Taken together, our findings indicate the feasibility of alphavirus-derived VLVs strategy in combating infection of filoviruses represented by EBOV and MARV, which provides further evidence of the potential of this platform for universal live-attenuated vaccine development.
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West Nile virus (WNV), an arthropod-borne flavivirus, can cause severe symptoms, including encephalitis, and death, posing a threat to public health and the economy. However, there is still no approved treatment or vaccine available for humans. Here, we developed a novel vaccine platform based on a classical insect-specific flavivirus (cISF) YN15-283-02, which was derived from Culicoides. The cISF-WNV chimera was constructed by replacing prME structural genes of the infectious YN15-283-02 cDNA clone with those of WNV and successfully rescued in Aedes albopictus cells. cISF-WNV was nonreplicable in vertebrate cells and nonpathogenic in type I interferon receptor (IFNAR)-deficient mice. A single-dose immunization of cISF-WNV elicited considerable Th1-biased antibody responses in C57BL/6 mice, which was sufficient to offer complete protection against lethal WNV challenge with no symptoms. Our studies demonstrated the potential of the insect-specific cISF-WNV as a prophylactic vaccine candidate to prevent infection with WNV.
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Aedes , Flavivirus , Vacinas , Febre do Nilo Ocidental , Vírus do Nilo Ocidental , Animais , Camundongos , Humanos , Vírus do Nilo Ocidental/genética , Flavivirus/genética , Febre do Nilo Ocidental/prevenção & controle , Anticorpos Antivirais , Camundongos Endogâmicos C57BLRESUMO
The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus, which is highly pathogenic and classified as a biosafety level 3 (BSL-3) agent, has greatly threatened global health and efficacious antivirals are urgently needed. The high requirement of facilities to manipulate the live virus has limited the development of antiviral study. Here, we constructed a reporter replicon of SARS-CoV-2, which can be handled in a BSL-2 laboratory. The Renilla luciferase activity effectively reflected the transcription and replication levels of the replicon genome. We identified the suitability of the replicon in antiviral screening using the known inhibitors, and thus established the replicon-based high-throughput screening (HTS) assay for SARS-CoV-2. The application of the HTS assay was further validated using a few hit natural compounds, which were screened out in a SARS-CoV-2 induced cytopathic-effect-based HTS assay in our previous study. This replicon-based HTS assay will be a safe platform for SARS-CoV-2 antiviral screening in a BSL-2 laboratory without the live virus.
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Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Replicon/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Animais , Chlorocebus aethiops , Descoberta de Drogas , Ensaios de Triagem em Larga Escala/métodos , Humanos , Replicon/genética , SARS-CoV-2/genética , Células Vero , Replicação Viral/efeitos dos fármacos , Tratamento Farmacológico da COVID-19RESUMO
BACKGROUND: Urgent-start dialysis is a major problem for incident dialysis population. Urgent start on hemodialysis is associated with an increased risk of infectious or mechanical complications, and its mortality is equal to or higher than that of urgent start on peritoneal dialysis (PD). However, compared to patients starting PD in a planned setting, those on urgent-started PD have an increased risk of mechanical complications and lower technique survival. METHODS: In this study, 101 adult incident dialysis patients (≥18 years old) who underwent Tenckhoff catheter implantation were enrolled. All of the patients were grouped according to the urgent PD mode: the intermittent PD (IPD) or automatic PD (APD) group, and patients were followed for 1 year. The paired or independent t test was used to analyze the change of laboratory variables. Pearson chi-square test was applied to compare the short outcome between the 2 groups. RESULTS: When PD was treated for 7 days and 1 month, the APD group has the lower serum potassium and phosphorus levels than the IPD group. The incidence of catheter dysfunction was significantly lower in the APD group. The morbidity of infection associated with PD in the first year was lower in the APD group despite no significant difference existing. The technique survival and patient survival rate have no evident difference between the 2 groups. CONCLUSION: Compared to IPD, urgent start on APD could reduce the risk of mechanical complication, which could be considered a gentle, safe, and feasible alternative to urgent start on IPD.
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Assistência Ambulatorial/métodos , Cateteres de Demora/efeitos adversos , Falência Renal Crônica/terapia , Diálise Peritoneal/métodos , Complicações Pós-Operatórias/epidemiologia , Estudos de Viabilidade , Feminino , Humanos , Falência Renal Crônica/sangue , Falência Renal Crônica/mortalidade , Masculino , Pessoa de Meia-Idade , Diálise Peritoneal/instrumentação , Diálise Peritoneal/mortalidade , Fosfatos/sangue , Complicações Pós-Operatórias/etiologia , Período Pós-Operatório , Potássio/sangue , Estudos Retrospectivos , Taxa de Sobrevida , Fatores de Tempo , Resultado do TratamentoRESUMO
OBJECTIVE: To investigate moisture content and hygroscopicity of spray dry powder of Gubi compound's water extract obtained at different spray drying conditions and laying a foundation for spray drying process of Chinese herbal compound preparation. METHOD: In the paper, on the basis of single-factor experiments, the author choose inlet temperature, liquid density, feed rate, air flow rate as investigated factors. RESULT: The experimental absorption rate-time curve and scanning electron microscopy results showed that under different spray drying conditions the spray-dried powders have different morphology and different adsorption process. CONCLUSION: At different spray-dried conditions, the morphology and water content of the powder is different, these differences lead to differences in the adsorption process, at the appropriate inlet temperature and feed rate with a higher sample density and lower air flow rate, in the experimental system the optimum conditions is inlet temperature of 150 degrees C, feed density of 1.05 g x mL(-1), feed rate of 20 mL x min(-1) air flow rate of 30 m3 x h(-1).
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Dessecação/métodos , Medicamentos de Ervas Chinesas/química , Interações Hidrofóbicas e Hidrofílicas , Tamanho da Partícula , Pós/química , Temperatura , Água/análise , MolhabilidadeRESUMO
Hand, foot, and mouth disease (HFMD) is a common pediatric illness mainly caused by enteroviruses, which are important human pathogens. Currently, there are no available antiviral agents for the therapy of enterovirus infection. In this study, an excellent high-content antiviral screening system utilizing the EV-A71-eGFP reporter virus was developed. Using this screening system, we screened a drug library containing 1042 natural compounds to identify potential EV-A71 inhibitors. Fangchinoline (FAN), a bis-benzylisoquinoline alkaloid, exhibits potential inhibitory effects against various enteroviruses that cause HFMD, such as EV-A71, CV-A10, CV-B3 and CV-A16. Further investigations revealed that FAN targets the early stage of the enterovirus life cycle. Through the selection of FAN-resistant EV-A71 viruses, we demonstrated that the VP1 protein could be a potential target of FAN, as two mutations in VP1 (E145G and V258I) resulted in viral resistance to FAN. Our research suggests that FAN is an efficient inhibitor of EV-A71 and has the potential to be a broad-spectrum antiviral drug against human enteroviruses.
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Antivirais , Benzilisoquinolinas , Farmacorresistência Viral , Antivirais/farmacologia , Humanos , Benzilisoquinolinas/farmacologia , Farmacorresistência Viral/genética , Replicação Viral/efeitos dos fármacos , Enterovirus Humano A/efeitos dos fármacos , Enterovirus Humano A/genética , Avaliação Pré-Clínica de Medicamentos , Genes Reporter , Ensaios de Triagem em Larga Escala , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/antagonistas & inibidores , Enterovirus/efeitos dos fármacos , Enterovirus/genética , Linhagem Celular , Proteínas de Fluorescência Verde/genéticaRESUMO
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 âcells. 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.
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Alphavirus , Animais , Alphavirus/genética , Vírus da Floresta de Semliki/genética , Vírus da Floresta de Semliki/metabolismo , Antivirais/farmacologia , Antivirais/metabolismo , Genes Reporter , Ensaios de Triagem em Larga Escala , Linhagem Celular , Replicação ViralRESUMO
The Omicron variant is sweeping the world, which displays striking immune escape potential through mutations at key antigenic sites on the spike protein, making broad-spectrum SARS-CoV-2 prevention or therapeutical strategies urgently needed. Previously, we have reported a hACE2-targeting neutralizing antibody 3E8, which could efficiently block both prototype SARS-CoV-2 and Delta variant infections in prophylactic mouse models, having the potential of broad-spectrum to prevent SARS-CoV-2. However, preparation of monoclonal neutralizing antibodies is severely limited by the time-consuming process and the relative high cost. Here, we utilized a modified VEEV replicon with two subgenomic (sg) promoters engineered to express the light and heavy chains of the 3E8 mAb. The feasibility and protective efficacy of replicating mRNA encoding 3E8 against Omicron infection in the hamster were demonstrated through the lung targeting delivery with the help of VEEV-VRP. Overall, we developed a safe and cost-effective platform of broad-spectrum to prevent SARS-CoV-2 infection.
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COVID-19 , SARS-CoV-2 , Animais , Cricetinae , Camundongos , SARS-CoV-2/genética , COVID-19/prevenção & controle , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes , RNA Mensageiro , Glicoproteína da Espícula de Coronavírus/genética , Anticorpos AntiviraisRESUMO
Despite global vaccination efforts, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve and spread globally. Currently, the development of affordable vaccine against Omicron variant of concern (VOC) is necessary. Here, we assessed the safety and immunogenicity of a SARS-CoV-2 vaccine consisting of a live Newcastle disease virus vector expressing the spike (S) protein of Omicron BA.1 administrated intranasally (IN) or intramuscularly (IM) in Golden Syrian hamster model. Immunogenicity studies showed that the prime-boost regimen elicited high antibody titers and the modified S antigen (Sm-F) could induce robust antibody response in low dosage immunization through IN route. Sera of the immunized hamsters provided effective cross-neutralizing activity against different Omicron variants, the prototype and delta strains of SARS-CoV-2. Moreover, the vaccine could provide complete immunoprotection in hamsters against the Omicron BA.1 challenge by either intranasal or intramuscular immunization. Overall, our study provides an alternative nasal vaccine against the SARS-CoV-2 Omicron variants.
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Antígenos de Grupos Sanguíneos , COVID-19 , Vacinas , Animais , Cricetinae , Humanos , Vírus da Doença de Newcastle/genética , SARS-CoV-2 , Vacinas contra COVID-19 , COVID-19/prevenção & controle , Vacinação , Imunização , Mesocricetus , Glicoproteína da Espícula de Coronavírus/genética , Anticorpos Neutralizantes , Anticorpos AntiviraisRESUMO
The binding geometries, abilities and thermodynamic parameters for the intermolecular complexation of two water-soluble calixarenes, p-sulfonatocalix[4]arene (SC4A) and p-sulfonatocalix[5]arene (SC5A), with biguanidinium guests, metformin (MFM) and phenformin (PFM), were investigated by (1)H and 2D NMR spectroscopy, X-ray crystallography, and isothermal titration calorimetry (ITC). The obtained results show that biguanidinium guests are captured by calixarenes with the alkyl or aromatic portion immersed into the cavities and the guanidinium portion fixed at the upper-rims. At both acidic and neutral conditions, SC4A always presents stronger binding affinities to biguanidinium guests than SC5A. Moreover, SC4A prefers to include MFM rather than PFM. As a result, the binding selectivity of MFM is up to 44.7 times for the SC4A/SC5A hosts. The intrinsic relationship between binding structures and selectivities were comprehensively analyzed and discussed from the viewpoint of thermodynamics. Finally, the ITC measurements were further performed in phosphate buffer instead of aqueous solution, to examine the buffer effects, counterion effect, and the differences between thermodynamic and apparent association constants.
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COVID-19 caused by SARS-CoV-2 has posed a significant threat to global public health since its outbreak in late 2019. Although there are a few drugs approved for clinical treatment to combat SARS-CoV-2 infection currently, the severity of the ongoing global pandemic still urges the efforts to discover new antiviral compounds. As the viral spike (S) protein plays a key role in mediating virus entry, it becomes a potential target for the design of antiviral drugs against COVID-19. Here, we tested the antiviral activity of berbamine hydrochloride, a bis-benzylisoquinoline alkaloid, against SARS-CoV-2 infection. We found that berbamine hydrochloride could efficiently inhibit SARS-CoV-2 infection in different cell lines. Further experiments showed berbamine hydrochloride inhibits SARS-CoV-2 infection by targeting the viral entry into host cells. Moreover, berbamine hydrochloride and other bis-benzylisoquinoline alkaloids could potently inhibit S-mediated cell-cell fusion. Furthermore, molecular docking results implied that the berbamine hydrochloride could bind to the post fusion core of SARS-CoV-2 S2 subunit. Therefore, berbamine hydrochloride may represent a potential efficient antiviral agent against SARS-CoV-2 infection.
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Benzilisoquinolinas , Tratamento Farmacológico da COVID-19 , Antivirais/farmacologia , Benzilisoquinolinas/farmacologia , Humanos , Fusão de Membrana , Simulação de Acoplamento Molecular , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Internalização do VírusRESUMO
The lung is the prophylaxis target against SARS-CoV-2 infection, and neutralizing antibodies are a leading class of biological products against various infectious viral pathogen. In this study, we develop a safe and cost-effective platform to express neutralizing antibody in the lung with replicating mRNA basing on alphavirus replicon particle (VRP) delivery system, to prevent SARS-CoV-2 infections. First, a modified VEEV replicon with two subgenomic (sg) promoters was engineered to translate the light and heavy chains of antibody simultaneously, for expression and assembly of neutralizing anti-SARS-CoV-2 antibody CB6. Second, the feasibility and protective efficacy of replicating mRNA against SARS-CoV-2 infection were demonstrated through both in vitro and in vivo assays. The lung target delivery with the help of VRP system resulted in efficiently block SARS-CoV-2 infection with reducing viral titer and less tissue damage in the lung of mice. Overall, our data suggests that expressing neutralizing antibodies in the lungs with the help of self-replicating mRNA could potentially be a promising prophylaxis approach against SARS-CoV-2 infection.
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Alphavirus , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/terapia , Replicon , SARS-CoV-2/metabolismo , Animais , Anticorpos Neutralizantes/biossíntese , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/genética , COVID-19/genética , COVID-19/metabolismo , Chlorocebus aethiops , Cricetinae , Feminino , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , SARS-CoV-2/genética , Células VeroRESUMO
The genus Flavivirus comprises numerous emerging and re-emerging arboviruses causing human illness. Vaccines are the best approach to prevent flavivirus diseases. But pathogen diversities are always one of the major hindrances for timely development of new vaccines when confronting unpredicted flavivirus outbreaks. We used West Nile virus (WNV) as a model to develop a new live-attenuated vaccine (LAV), WNV-poly(A), by replacing 5' portion (corresponding to SL and DB domains in WNV) of 3'-UTR with internal poly(A) tract. WNV-poly(A) not only propagated efficiently in Vero cells, but also was highly attenuated in mouse model. A single-dose vaccination elicited robust and long-lasting immune responses, conferring full protection against WNV challenge. Such "poly(A)" vaccine strategy may be promising for wide application in the development of flavivirus LAVs because of its general target regions in flaviviruses.
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Febre do Nilo Ocidental , Vacinas contra o Vírus do Nilo Ocidental , Regiões 3' não Traduzidas , Animais , Anticorpos Antivirais , Chlorocebus aethiops , Camundongos , Poli A , Células Vero , Febre do Nilo Ocidental/prevenção & controleRESUMO
Japanese encephalitis virus (JEV), a major cause of Japanese encephalitisis, is an arbovirus that belongs to the genus Flavivirus of the family Flaviviridae. Currently, there is no effective drugs available for the treatment of JEV infection. Therefore, it is important to establish efficient antiviral screening system for the development of antiviral drugs. In this study, we constructed a full-length infectious clone of eGFP-JEV reporter virus by inserting the eGFP gene into the capsid-coding region of the viral genome. The reporter virus RNA transfected-BHK-21 cells generated robust eGFP fluorescence signals that were correlated well with viral replication. The reporter virus displayed growth kinetics similar to wild type (WT) virus although replicated a little slower. Using a known JEV inhibitor, NITD008, we demonstrated that the reporter virus could be used to identify inhibitors against JEV. Furthermore, an eGFP-JEV-based high throughput screening (HTS) assay was established in a 96-well format and used for screening of 1443 FDA-approved drugs. Sixteen hit drugs were identified to be active against JEV. Among them, five compounds which are lonafarnib, cetylpyridinium chlorid, cetrimonium bromide, nitroxoline and hexachlorophene, are newly discovered inhibitors of JEV, providing potential new therapies for treatment of JEV infection.
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Vírus da Encefalite Japonesa (Espécie)/efeitos dos fármacos , Vírus da Encefalite Japonesa (Espécie)/genética , Genes Reporter , Proteínas de Fluorescência Verde/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Cricetinae , Culicidae , Avaliação Pré-Clínica de Medicamentos , Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Rim/citologia , Estados Unidos , United States Food and Drug Administration , Replicação Viral/efeitos dos fármacosAssuntos
Modelos Animais de Doenças , Animais , Camundongos , Vacinas Virais/imunologia , Vacinas Virais/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/administração & dosagem , Vesiculovirus/genética , Vesiculovirus/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologiaRESUMO
BACKGROUND: Peritonitis, which is one of the leading complications of peritoneal dialysis (PD) worldwide, severely affected morbidity and mortality of the PD patients. Although many efforts have been made to prevent PD-related peritonitis, it seems impossible to prevent it completely. Many causes have been reported to lead to peritonitis, and contamination during bag exchange is one of the important risk factors for peritonitis. METHODS: Here, we introduce an operating vehicle, which we invented to provide a sterile and safe space for bag exchange. A single-center, retrospective, case-control study was undertaken to determine whether this operating vehicle has a protective role in preventing peritonitis. In total, 462 continuous ambulatory peritoneal dialysis patients were included in this study from October 2014 to March 2017. According to their personal will, these patients chose to use operating vehicle or traditional method during their bag exchange. The demographic, clinical and laboratory data of these patients in the two groups were collected, analyzed and compared. RESULTS: Of 462 patients with home dialysis, operating vehicle group consisted of 61 patients, and control group consisted of 401 patients. In the control group, over 677 patient-years, peritonitis occurred in 69 of 401 patients (17.2%), while in the operating vehicle group, over 60 patient-years, only 4 of 61 patients (6.6%) had episodes of peritonitis. The number of patients suffered from peritonitis was significantly decreased in the operating vehicle group (P = 0.034). Besides, there were a total of 99 episodes of peritonitis, and the rate was 1 episode every 7.2 patient-years in control group and 1 episode every 12 patient-years in the operating vehicle group. There was significant difference between the two groups (0.013). Positive dialysate cultures were obtained in majority of the peritonitis episodes (60.6%). CONCLUSION: Operating vehicle might help to reduce PD-related peritonitis by preventing contamination during bag exchange. Further studies are still needed to demonstrate the protective role of the operating vehicle in preventing peritonitis.