RESUMO
The method of ultra-high performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry(UHPLC-Q/Orbitrap HRMS)combined with molecular network was developed in this study for rapidly analyzing the chemical components of the Qinggu San reference sample of classical prescription. Firstly, an ACQUITY UPLC BEH Shield RP_(18) column(2.1 mm×100 mm, 1.7 µm)was used, and acetonitrile and 0.1% formic acid were taken as the mobile phases for gradient elution. The flow rate was 0.4 mL·min~(-1), and the column temperature was 30 â. Under these conditions, the mass spectrum data were collected in both positive and negative ion modes of the heated electrospray ionization source. Subsequently, the mass spectrum data of the Qinggu San reference sample were uploaded to the Global Natural Products Social Molecular Network(GNPS)platform for calculation and analysis, and a visual molecular network was built with Cytoscape 3.8.2 software. On this basis, the chemical components of the Qinggu San reference sample were identified by fragmentation regularity of standard compounds, retention time, accurate relative molecular weight of HR-MS, characteristic fragment ions information, literature, and databases. Finally, a total of 105 chemical components were identified and speculated in the Qinggu San reference sample, including 19 iridoid glycosides, 23 flavonoids, 15 phenylpropanoids, 11 triterpene saponins, and 37 other components. Meanwhile, two of these components are potential new compounds. The method used in this study not only achieved rapid and accurate identification of chemical components in the Qinggu San reference sample and provided a scie-ntific basis for the study of pharmacological substances and quality control of Qinggu San compound preparations but also provided a refe-rence for the rapid identification of chemical components in traditional Chinese medicine compound preparations.
Assuntos
Medicamentos de Ervas Chinesas , Cromatografia Líquida de Alta Pressão/métodos , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/análise , Espectrometria de Massas/métodosRESUMO
Rabies is a zoonotic neurological disease caused by the rabies virus (RABV) that is fatal to humans and animals. While several post-infection treatment have been suggested, developing more efficient and innovative antiviral methods are necessary due to the limitations of current therapeutic approaches. To address this challenge, a strategy combining photodynamic therapy and immunotherapy, using a photosensitizer (TPA-Py-PhMe) with high type I and type II reactive oxygen species (ROS) generation ability is proposed. This approach can inactivate the RABV by killing the virus directly and activating the immune response. At the cellular level, TPA-Py-PhMe can reduce the virus titer under preinfection prophylaxis and postinfection treatment, with its antiviral effect mainly dependent on ROS and pro-inflammatory factors. Intriguingly, when mice are injected with TPA-Py-PhMe and exposed to white light irradiation at three days post-infection, the onset of disease is delayed, and survival rates improved to some extent. Overall, this study shows that photodynamic therapy and immunotherapy open new avenues for future antiviral research.
Assuntos
Fotoquimioterapia , Vírus da Raiva , Raiva , Humanos , Animais , Camundongos , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Espécies Reativas de Oxigênio , Raiva/prevenção & controle , Raiva/tratamento farmacológico , AntiviraisRESUMO
The ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was used to conduct the qualitative analysis of the monoterpene chemical components from Paeoniae Radix Rubra. Gradient elution was performed on C_(18) HD(2.1 mm×100 mm, 2.5 µm) column with a mobile phase of 0.1% formic acid(A) and acetonitrile(B). The flow rate was 0.4 mL·min~(-1) and the column temperature was 30 â. MS analysis was conducted in both positive and negative ionization modes using electrospray ionization(ESI) source. Qualitative Analysis 10.0 was used for data processing. The identification of chemical components was realized by the combination of standard compounds, fragmentation patterns, and mass spectra data reported in the literature. Forty-one monoterpenoids in Paeoniae Radix Rubra extract were identified. Among them, 8 compounds were reported in Paeoniae Radix Rubra for the first time and 1 was presumed to be the new compound 5â³-O-methyl-galloylpaeoniflorin or its positional isomer. The method in this study realizes the rapid identification of monoterpenoids from Paeoniae Radix Rubra and provides a material and scientific basis for quality control and further study on the pharmaceutical effect of Paeoniae Radix Rubra.
Assuntos
Medicamentos de Ervas Chinesas , Cromatografia Líquida , Espectrometria de Massas , MonoterpenosRESUMO
Qingfei Paidu Decoction is a Chinese medicine formula that has been proved effective in the treatment of coronavirus disease 2019. However, the comprehensive separation and characterization of Qingfei Paidu Decoction are of a great challenge due to the diversity of chemical components in a wide range of polarity. In this study, a triplex off-line two-dimensional liquid chromatography coupled with quadrupole time-of-flight mass spectrometry is developed for the analysis of Qingfei Paidu Decoction. One reversed-phase liquid chromatography×hydrophilic interaction liquid chromatography system and two reversed-phase liquid chromatography×reversed phase liquid chromatography systems were constructed to separate polar components and weak-polar components in Qingfei Paidu Decoction, respectively. Benefiting from the good orthogonality of two-dimensional liquid chromatography and high sensitivity of quadrupole time-of-flight MS, chemical components with different polarities and content were discovered. A total of 749 peaks were detected in positive and negative ionization mode and presented as a four-dimensional data plot. Meanwhile, 498 compounds belonging to 14 categories were tentatively identified. These results provide good supplementary to elucidate the material basis of Qingfei Paidu Decoction. The triplex off-line two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry strategy can be a powerful and efficient tool for the separation and characterization of chemical substances in traditional Chinese medicine formulas.
Assuntos
COVID-19 , Medicamentos de Ervas Chinesas , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida , Medicamentos de Ervas Chinesas/análise , Humanos , Espectrometria de Massas/métodosRESUMO
The breadth and depth of traditional Chinese medicine (TCM) applications have been expanding in recent years, yet the problem of quality control has arisen in the application process. It is essential to design an algorithm to provide blending ratios that ensure a high overall product similarity to the target with controlled deviations in individual ingredient content. We developed a new blending algorithm and scheme by comparing different samples of ginkgo leaves. High-consistency samples were used to establish the blending target, and qualified samples were used for blending. Principal component analysis (PCA) was used as the sample screening method. A nonlinear programming algorithm was applied to calculate the blending ratio under different blending constraints. In one set of calculation experiments, the result was blended by the same samples under different conditions. Its relative deviation coefficients (RDCs) were controlled within ±10%. In another set of calculations, the RDCs of more component blending by different samples were controlled within ±20%. Finally, the near-critical calculation ratio was used for the actual experiments. The experimental results met the initial setting requirements. The results show that our algorithm can flexibly control the content of TCMs. The quality control of the production process of TCMs was achieved by improving the content stability of raw materials using blending. The algorithm provides a groundbreaking idea for quality control of TCMs.
Assuntos
Medicamentos de Ervas Chinesas , Ginkgo biloba , Cromatografia Líquida de Alta Pressão , Medicamentos de Ervas Chinesas/análise , Medicina Tradicional Chinesa , Folhas de Planta/química , Controle de QualidadeRESUMO
Ginseng, which contains abundant ginsenosides, grows mainly in the Jilin, Liaoning, and Heilongjiang in China. It has been reported that the quality and traits of ginsengs from different origins were greatly different. To date, the accurate prediction of the origins of ginseng samples is still a challenge. Here, we integrated ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) with a support vector machine (SVM) for rapid discrimination and prediction of ginseng from the three main regions where it is cultivated in China. Firstly, we develop a stable and reliable UHPLC-Q-TOF-MS method to obtain robust information for 31 batches of ginseng samples after reasonable optimization. Subsequently, a rapid pre-processing method was established for the rapid screening and identification of 69 characteristic ginsenosides in 31 batches ginseng samples from three different origins. The SVM model successfully distinguished ginseng origin, and the accuracy of SVM model was improved from 83% to 100% by optimizing the normalization method. Six crucial quality markers for different origins of ginseng were screened using a permutation importance algorithm in the SVM model. In addition, in order to validate the method, eight batches of test samples were used to predict the regions of cultivation of ginseng using the SVM model based on the six selected quality markers. As a result, the proposed strategy was suitable for the discrimination and prediction of the origin of ginseng samples.
Assuntos
Ginsenosídeos , Panax , Biomarcadores , Cromatografia Líquida de Alta Pressão/métodos , Ginsenosídeos/química , Espectrometria de Massas/métodos , Panax/química , Máquina de Vetores de SuporteRESUMO
Ebola virus (EBOV) infections result in aggressive hemorrhagic fever in humans, with fatality rates reaching 90% and with no licensed specific therapeutics to treat ill patients. Advances over the past 5 years have firmly established monoclonal antibody (MAb)-based products as the most promising therapeutics for treating EBOV infections, but production is costly and quantities are limited; therefore, MAbs are not the best candidates for mass use in the case of an epidemic. To address this need, we generated EBOV-specific polyclonal F(ab')2 fragments from horses hyperimmunized with an EBOV vaccine. The F(ab')2 was found to potently neutralize West African and Central African EBOV in vitro Treatment of nonhuman primates (NHPs) with seven doses of 100 mg/kg F(ab')2 beginning 3 or 5 days postinfection (dpi) resulted in a 100% survival rate. Notably, NHPs for which treatment was initiated at 5 dpi were already highly viremic, with observable signs of EBOV disease, which demonstrated that F(ab')2 was still effective as a therapeutic agent even in symptomatic subjects. These results show that F(ab')2 should be advanced for clinical testing in preparation for future EBOV outbreaks and epidemics.IMPORTANCE EBOV is one of the deadliest viruses to humans. It has been over 40 years since EBOV was first reported, but no cure is available. Research breakthroughs over the past 5 years have shown that MAbs constitute an effective therapy for EBOV infections. However, MAbs are expensive and difficult to produce in large amounts and therefore may only play a limited role during an epidemic. A cheaper alternative is required, especially since EBOV is endemic in several third world countries with limited medical resources. Here, we used a standard protocol to produce large amounts of antiserum F(ab')2 fragments from horses vaccinated with an EBOV vaccine, and we tested the protectiveness in monkeys. We showed that F(ab')2 was effective in 100% of monkeys even after the animals were visibly ill with EBOV disease. Thus, F(ab')2 could be a very good option for large-scale treatments of patients and should be advanced to clinical testing.
Assuntos
Anticorpos Neutralizantes/imunologia , Vacinas contra Ebola/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Fragmentos Fab das Imunoglobulinas/imunologia , Macaca mulatta/virologia , Animais , Anticorpos Antivirais/imunologia , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/veterinária , Cavalos/imunologia , Imunização , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Imunoterapia/métodosRESUMO
Mink enteritis virus (MEV) is a parvovirus that causes acute enteritis in mink. The capsid protein VP2 of MEV is a major immunogenicity that is important for disease prevention. In this study, this protein was expressed in Spodoptera frugiperda 9 cells using a recombinant baculovirus system and was observed to self-assemble into virus-like particles (VLPs) with a high hemagglutination (HA) titer (1:216). A single-dose injection of VLPs (HA titer, 1:256) resulted in complete protection of mink against virulent MEV challenge for at least 180 days. These data suggest that these MEV VLPs could be used as a vaccine for the prevention of viral enteritis in mink.
Assuntos
Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Enterite Viral do Vison/prevenção & controle , Vírus da Enterite do Vison/imunologia , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Proteínas do Capsídeo/administração & dosagem , Expressão Gênica , Vison/imunologia , Vison/virologia , Enterite Viral do Vison/imunologia , Enterite Viral do Vison/virologia , Vírus da Enterite do Vison/genética , Vírus da Enterite do Vison/patogenicidade , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Células Sf9 , Spodoptera , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vacinas Virais/imunologia , VirulênciaRESUMO
This study is to explore the effect of Qingfei Paidu Decoction(QPD) on the host metabolism and gut microbiome of rats with metabolomics and 16 S rDNA sequencing. Based on 16 S rDNA sequencing of gut microbiome and metabolomics(GC-MS and LC-MS/MS), we systematically studied the serum metabolites profile and gut microbiota composition of rats treated with QPD for continued 5 days by oral gavage. A total of 23 and 43 differential metabolites were identified based on QPD with GC-MS and LC-MS/MS, respectively. The involved metabolic pathways of these differential metabolites included glycerophospholipid metabolism, linoleic acid metabolism, TCA cycle and pyruvate metabolism. Meanwhile, we found that QPD significantly regulated the composition of gut microbiota in rats, such as enriched Romboutsia, Turicibacter, and Clostridium_sensu_stricto_1, and decreased norank_f_Lachnospiraceae. Our current study indicated that short-term intervention of QPD could significantly regulate the host metabolism and gut microbiota composition of rats dose-dependently, suggesting that the clinical efficacy of QPD may be related with the regulation on host metabolism and gut microbiome.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Animais , Bactérias/classificação , Cromatografia Líquida , Metabolômica , Ratos , Espectrometria de Massas em TandemRESUMO
Japanese encephalitis virus SA14-14-2 (JEV SA14-14-2) is a widely used vaccine in China and other southeastern countries to prevent Japanese encephalitis in children. In this study, a stable infectious cDNA clone of JEV SA14-14-2 with a low copy number pACYC177 vector dependent on the T7 promoter and T7 terminator was developed. Two introns were inserted into the capsid gene and envelope gene of JEV cDNA for gene stability. Hepatitis delta virus ribozyme (HDVr) was engineered into the 3' UTR cDNA of JEV for authentic 3' UTR transcription. The rescued virus showed biological properties indistinguishable from those of the parent strain (JEV SA14-14-2). The establishment of a JEV SA14-14-2 reverse genetics system lays the foundation for the further development of other flavivirus vaccines and viral pathogenesis studies.
Assuntos
Vírus da Encefalite Japonesa (Espécie)/genética , Genética Reversa/métodos , Linhagem Celular , DNA Complementar , DNA Viral , Vírus da Encefalite Japonesa (Espécie)/ultraestrutura , Vetores Genéticos , Genoma Viral , Regiões Promotoras Genéticas , Sequenciamento do ExomaRESUMO
Scopolia tangutica is a traditional Chinese medicine used for antispasmodic, anesthesia, analgesia, and sedation. Its medicinal activity is associated to alkaloid constituents, including tropane and cinnamamide types. Low content of alkaloids in plant makes them difficult to be isolated and identified. The present work developed an effective method to quickly characterize alkaloids from Scopolia tangutica by high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry. Thirteen reference compounds were studied for their fragmentation pathways, including five tropane alkaloids and eight cinnamamide ones. Alkaloid constituent was analyzed by an optimized high-performance liquid chromatography method and mass spectrometry analysis to achieve systematic characterization of alkaloids from Scopolia tangutica. As a result, 53 compounds were identified, including 21 tropane alkaloids (eight new ones), 18 caffeoyl ones (ten new ones) and 14 dicaffeoyl ones (seven new ones). It was important to provide rich information in phytochemical study and structure-guided isolation of important compounds from this plant.
Assuntos
Alcaloides/isolamento & purificação , Cinamatos/isolamento & purificação , Medicamentos de Ervas Chinesas/isolamento & purificação , Extratos Vegetais/isolamento & purificação , Scopolia/química , Tropanos/isolamento & purificação , Alcaloides/química , Cromatografia Líquida de Alta Pressão , Cinamatos/química , Medicamentos de Ervas Chinesas/química , Medicina Tradicional Chinesa , Extratos Vegetais/química , Espectrometria de Massas em Tandem , Fatores de Tempo , Tropanos/químicaRESUMO
Porcine epidemic diarrhea virus (PEDV), which causes 80-100% mortality in neonatal piglets, is one of the most devastating viral diseases affecting swine worldwide. To date, the lack of effective vaccines and drugs is the main problem preventing control of the global spread of PEDV. In this study, we produced PEDV virus-like particles (VLPs) composed of S, M, and E proteins with a baculovirus expression system and tested them via indirect immunofluorescence assay (IFA)and Western blot analysis. Electron microscopy showed that the morphological structure of the PEDV VLPs was similar to that of the protovirus. Microneutralization assays and ELISpot analysis demonstrated that PEDV VLPs induced highly specific antibody responses and Th2-mediated humoral immunity. As a result, the PEDV VLPs displayed excellent immunogenicity in mice. Therefore, a VLP-based vaccine has the potential to prevent PEDV infection.
Assuntos
Infecções por Coronavirus/veterinária , Imunidade Humoral , Vírus da Diarreia Epidêmica Suína/imunologia , Doenças dos Suínos/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Vírus da Diarreia Epidêmica Suína/genética , Suínos , Doenças dos Suínos/prevenção & controle , Doenças dos Suínos/virologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vacinas Virais/imunologiaRESUMO
An optimized VP2 gene from the current prevalent CPV strain (new CPV-2a) in China was expressed in a baculovirus expression system. It was found that the VP2 proteins assembled into virus-like particles (VLPs) with antigenic properties similar to those of natural CPV and with an especially high hemagglutination (HA) titer (1:2(20)). Dogs intramuscularly or orally immunized with VLPs produced antibodies against CPV with >1:80 hemagglutination inhibition (HI) units for at least 3 months. The CPV VLPs could be considered for use as a vaccine against CPV or as a platform for research on chimeric VLP vaccines against other diseases.
Assuntos
Baculoviridae , Vetores Genéticos , Parvovirus Canino/genética , Proteínas Estruturais Virais/metabolismo , Virossomos/metabolismo , Administração Oral , Animais , Anticorpos Antivirais/sangue , China , Cães , Injeções Intramusculares , Multimerização Proteica , Vacinas Virossomais/administração & dosagem , Vacinas Virossomais/genética , Vacinas Virossomais/imunologia , Proteínas Estruturais Virais/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vacinas Virais/imunologia , Virossomos/genéticaRESUMO
Canine parvovirus (CPV) can cause severe disease in animals and continuously generates new variant and recombinant strains in dogs that have a strong impact on sanitation. It is therefore necessary to investigate epidemic CPV strains to improve our understanding of CPV transmission and epidemic behavior. However, most studies have focused on the analysis of VP2, and therefore, information about recombination and relationships between strains is still lacking. Here, 14 strains of CPV were isolated from domestic dogs suspected of hosting CPV between 2013 and 2014 in China. The complete NS1 and VP2 genes were sequenced and analyzed. The results suggest that the new CPV-2a and new CPV-2b types are the prevalent strains in China. In addition to a few mutations (residues 19, 544, 545, 572 and 583 of NS1 and residues 267, 370, 377 and 440 of VP2) that were preserved during transmission, new mutations (residues 60, 630 of NS1, and residues 21, 310 of VP2) were found in the isolated strains. A phylogenetic tree based on VP2 sequences illustrated that the new CPV-2a and new CPV-2b strains from China form single clusters that are distinct from lineages from other countries. Moreover, recombination between the new CPV-2a and new CPV-2b types was also identified in the isolated strains. Due to differences in selection pressures or recombination, there were a small number of inconsistencies between the phylogenetic trees for VP2 and NS1, which indicated that phylogenetic relationships based on VP2 might not be representative of those based on NS1. The data indicated that mutations and recombination are constantly occurring along with the spread of CPV in China.
Assuntos
Doenças do Cão/virologia , Infecções por Parvoviridae/veterinária , Parvovirus Canino/genética , Parvovirus Canino/isolamento & purificação , Animais , China/epidemiologia , Análise por Conglomerados , DNA Viral/química , DNA Viral/genética , Doenças do Cão/epidemiologia , Cães , Variação Genética , Epidemiologia Molecular , Dados de Sequência Molecular , Mutação , Infecções por Parvoviridae/epidemiologia , Infecções por Parvoviridae/virologia , Filogenia , Recombinação Genética , Análise de Sequência de DNA , Homologia de Sequência , Proteínas Virais/genéticaRESUMO
Rabies virus infection is a major public health concern because of its wide host-interference spectrum and nearly 100 % lethality. However, the interactions between host and virus remain unclear. To decipher the authentic response in the central nervous system after rabies virus infection, a dynamic analysis of brain proteome alteration was performed. In this study, 104 significantly differentially expressed proteins were identified, and intermediate filament, interferon-inducible GTPases, and leucine-rich repeat-containing protein 16C were the three outstanding groups among these proteins. Interferon-inducible GTPases were prominent because of their strong upregulation. Moreover, quantitative real-time PCR showed distinct upregulation of interferon-inducible GTPases at the level of transcription. Several studies have shown that interferon-inducible GTPases are involved in many biological processes, such as viral infection, endoplasmic reticulum stress response, and autophagy. These findings indicate that interferon-inducible GTPases are likely to be a potential target involved in rabies pathogenesis or the antiviral process.
Assuntos
GTP Fosfo-Hidrolases/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Interferons/fisiologia , Raiva/metabolismo , Animais , Química Encefálica , Feminino , GTP Fosfo-Hidrolases/análise , Camundongos , Camundongos Endogâmicos BALB C , Proteômica/métodos , Vírus da Raiva/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas em TandemRESUMO
Ebola virus (species Zaire ebolavirus) (EBOV) is highly virulent in humans. The largest recorded outbreak of Ebola hemorrhagic fever in West Africa to date was caused by EBOV. Therefore, it is necessary to develop a detection method for this virus that can be easily distributed and implemented. In the current study, we developed a visual assay that can detect EBOV-associated nucleic acids. This assay combines reverse transcription loop-mediated isothermal amplification and nucleic acid strip detection (RT-LAMP-NAD). Nucleic acid amplification can be achieved in a one-step process at a constant temperature (58 °C, 35 min), and the amplified products can be visualized within 2-5 min using a nucleic acid strip detection device. The assay is capable of detecting 30 copies of artificial EBOV glycoprotein (GP) RNA and RNA encoding EBOV GP from 10(2) TCID50 recombinant viral particles per ml with high specificity. Overall, the RT-LAMP-NAD method is simple and has high sensitivity and specificity; therefore, it is especially suitable for the rapid detection of EBOV in African regions.
Assuntos
Ebolavirus , Doença pelo Vírus Ebola/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Ebolavirus/genética , Humanos , RNA Viral/genética , RNA Viral/isolamento & purificação , Transcrição Reversa , Sensibilidade e Especificidade , Alinhamento de SequênciaRESUMO
Rabies virus (RABV) can cause a fatal neurological disease in human and animals, and vaccines were generally applied for the immunoprophylaxis of rabies. Here, a recombinant viral vector carrying the exogenous gene expression component between phosphoprotein (P) and matrix protein (M) genes of RABV was constructed based on the vaccine strain SRV9 used in China. To develop a reverse genetic system, the full-length cDNA plasmids of SRV9 were constructed using the eukaryotic expression vector pCI or pcDNA3.1(+). However, recovery efficiency based on the pcDNA3.1 vector was significantly higher than that of the pCI vector. The exogenous gene expression component PE-PS-BsiWI-PmeI or PS-BsiWI-PmeI-PE was introduced in different locations between the P and M genes of SRV9. When the enhanced green fluorescent protein (eGFP) was used as a reporter gene, both locations could rescue recombinant RABV (rRABV) expressing eGFP with high efficiency. Characterization of rRABV expressing eGFP in vitro revealed that its growth was similar to that of the parental virus. Animal experiments showed that rRABV expressing eGFP could replicate and express eGFP in the brains of suckling mice. Furthermore, rRABV of SRV9 was nonpathogenic for 3-week-old mice and could be cleared from the central nervous system at 5 days post-inoculation. Our results showed that the recombinant SRV9 virus could be used as a useful viral vector for exogenous gene expression.
Assuntos
Expressão Gênica , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Vacina Antirrábica/genética , Vírus da Raiva/genética , Animais , Feminino , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/genética , Plasmídeos/metabolismo , Vacina Antirrábica/metabolismo , Vírus da Raiva/metabolismo , Genética ReversaRESUMO
Feline viral rhinotracheitis (FVR) is caused by the feline herpesvirus-1 (FHV-1), which commonly results in upper respiratory symptoms, and can result in death in the kittens and weak cats. Rabies is an infectious disease with zoonotic characteristics highly relevant to public health and also poses a serious threat to cats. Vaccines are the most effective method to control the spread of both FHV-1 and RABV and have the advantage that they produce long-term specific immune responses. In this study, we constructed a bivalent vaccine against FHV-1 and rabies virus (RABV) simultaneously. The vaccine was constructed by cloning FHV-1 gB into a RABV based vector, and the recombinant RABV (SRV9-FHV-gB) expressing the FHV-1 gB protein was rescued. The growth characteristics of SRV9-FHV-gB were analyzed on NA and BSR cells. To assess the immunogenicity of the vaccine, mice and cats were immunized with SRV9-FHV-gB supplemented with Gel02 adjuvant. The SRV9-FHV-gB exhibited the same growth characteristics as the parent virus SRV9 in both BSR cells and NA cells. The safety of SRV9-FHV-gB was evaluated using 5-day-old and 14-day-old suckling mice. The results showed that mice infected with the SRV9-FHV-gB survived for longer than those in the SRV9 group. Mice immunized with inactivated SRV9-FHV-gB produced high titers of specific antibodies against FHV-1 and neutralizing antibodies against RABV. Cats that received three immunizations with SRV9-FHV-gB also produced neutralizing antibodies against both FHV-1 and RABV. This study represents the first time that a bivalent vaccine targeting FHV-1 and RABV has been constructed, laying the foundations and providing inspiration for the development of other multivalent vaccines.
Assuntos
Doenças do Gato , Vacina Antirrábica , Vírus da Raiva , Raiva , Doenças dos Roedores , Varicellovirus , Gatos , Animais , Feminino , Camundongos , Raiva/prevenção & controle , Raiva/veterinária , Vírus da Raiva/genética , Vacinas Combinadas , Vacinas Sintéticas , Anticorpos Neutralizantes , Anticorpos Antivirais , Doenças do Gato/prevenção & controleRESUMO
The rapid emergence of Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2) variants, coupled with severe immune evasion and imprinting, has jeopardized the vaccine efficacy, necessitating urgent development of broad protective vaccines. Here, we propose a strategy employing recombinant rabies viruses (RABV) to create a universal SARS-CoV-2 vaccine expressing heterologous tandem receptor-binding domain (RBD) trimer from the SARS-CoV-2 Prototype, Delta, and Omicron strains (SRV-PDO). The results of mouse immunization indicated that SRV-PDO effectively induced cellular and humoral immune responses, and demonstrated higher immunogenicity and broader SARS-CoV-2 neutralization compared to the recombinant RABVs that only expressed RBD monomers. Moreover, SRV-PDO exhibited full protection against SARS-CoV-2 in the challenge assay. This study demonstrates that recombinant RABV expressing tandem RBD-heterotrimer as a multivalent immunogen could elicit a broad-spectrum immune response and potent protection against SARS-CoV-2, making it a promising candidate for future human or veterinary vaccines and offering a novel perspective in other vaccine design.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Vacinas contra COVID-19 , COVID-19 , Camundongos Endogâmicos BALB C , Vírus da Raiva , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Animais , Vírus da Raiva/imunologia , Vírus da Raiva/genética , Vacinas contra COVID-19/imunologia , Camundongos , SARS-CoV-2/imunologia , SARS-CoV-2/genética , COVID-19/prevenção & controle , COVID-19/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , Feminino , Humanos , Imunidade Humoral , Vetores Genéticos , Eficácia de Vacinas , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/administração & dosagemRESUMO
Purification of high-purity compounds from traditional Chinese medicines (TCMs) plays an important role in investigating their bioactivity. Nevertheless, it is often quite difficult to isolate compounds with high purity because of the complexity of TCMs in chemical composition. In this work, a two-dimensional preparation method was successfully developed for the preparation of high-purity compounds from the stem of Lonicera japonica Thunb, based on two novel polar copolymerized RP stationary phases, XAqua C3 and XAqua C18. An XAqua C3 prep column was used to separate the sample in the first-dimensional preparation, and 14 g of sample was fractionated into eight fractions with a recovery of 82%. An XAqua C18 prep column was selected to prepare high-purity compounds in the second-dimensional preparation for its good orthogonality with the XAqua C3 stationary phase. As a result, major compounds in the sample were isolated with more than 99% purity. This method is a potent method to realize the efficient purification of compounds with high purity from the stem of L. japonica Thunb and it shows great potential in the separation of high-purity compounds from complex samples.