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COVID-19 is currently pandemic and the detection of SARS-CoV-2 variants in wastewater is causing widespread concern. Herein, cold atmospheric plasma (CAP) is proposed as a novel wastewater disinfection technology that effectively inactivates SARS-CoV-2 transcription- and replication-competent virus-like particles, coronavirus GX_P2V, pseudotyped SARS-CoV-2 variants, and porcine epidemic diarrhoea virus in a large volume of water within 180 s (inhibition rate > 99%). Further, CAP disinfection did not adversely affect the viability of various human cell lines. It is identified that CAP produced peroxynitrite (ONOO-), ozone (O3), superoxide anion radicals (O2 -), and hydrogen peroxide (H2O2) as the major active substances for coronavirus disinfection. Investigation of the mechanism showed that active substances not only reacted with the coronavirus spike protein and affected its infectivity, but also destroyed the nucleocapsid protein and genome, thus affecting virus replication. This method provides an efficient and environmentally friendly strategy for the elimination of SARS-CoV-2 and other coronaviruses from wastewater.
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BACKGROUND: The monkeypox virus (MPXV) is a linear double-stranded DNA virus with a large genome that causes tens of thousands of infections and hundreds of deaths in at least 40 countries and regions worldwide. Therefore, timely and accurate diagnostic testing could be an important measure to prevent the ongoing spread of MPXV and widespread epidemics. RESULTS: Here, we designed multiple sets of primers for the target region of MPXV for loop-mediated isothermal amplification (LAMP) detection and identified the optimal primer set. Then, the specificity in fluorescent LAMP detection was verified using the plasmids containing the target gene, pseudovirus and other DNA/RNA viruses. We also evaluated the sensitivity of the colorimetric LAMP detection system using the plasmid and pseudovirus samples, respectively. Besides, we used monkeypox pseudovirus to simulate real samples for detection. Subsequent to the establishment and introduction of a magnetic beads (MBs)-based nucleic acid extraction technique, an integrated device was developed, characterized by rapidity, high sensitivity, and remarkable specificity. This portable system demonstrated a visual detection limit of 137 copies/mL, achieving sample-to-answer detection within 1 h. SIGNIFICANCE: The device has the advantages of integration, simplicity, miniaturization, and visualization, which help promote the realization of accurate, rapid, portable, and low-cost testing. Meanwhile, this platform could facilitate efficient, cost-effective and easy-operable point-of-care testing (POCT) in diverse resource-limited settings in addition to the laboratory.
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Colorimetría , Monkeypox virus , Técnicas de Amplificación de Ácido Nucleico , Colorimetría/métodos , Colorimetría/instrumentación , Técnicas de Amplificación de Ácido Nucleico/métodos , Monkeypox virus/genética , Monkeypox virus/aislamiento & purificación , Límite de Detección , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/instrumentaciónRESUMEN
Menispermaceae species, as early-diverging eudicots, can synthesize valuable benzylisoquinoline alkaloids (BIAs) like bisbenzylisoquinoline alkaloids (bisBIAs) and sinomenines with a wide range of structural diversity. However, the evolutionary mechanisms responsible for their chemo-diversity are not well understood. Here, a chromosome-level genome assembly of Menispermum dauricum is presented and demonstrated the occurrence of two whole genome duplication (WGD) events that are shared by Ranunculales and specific to Menispermum, providing a model for understanding chromosomal evolution in early-diverging eudicots. The biosynthetic pathway for diverse BIAs in M. dauricum is reconstructed by analyzing the transcriptome and metabolome. Additionally, five catalytic enzymes - one norcoclaurine synthase (NCS) and four cytochrome P450 monooxygenases (CYP450s) - from M. dauricum are responsible for the formation of the skeleton, hydroxylated modification, and C-O/C-C phenol coupling of BIAs. Notably, a novel leaf-specific MdCYP80G10 enzyme that catalyzes C2'-C4a phenol coupling of (S)-reticuline into sinoacutine, the enantiomer of morphinan compounds, with predictable stereospecificity is discovered. Moreover, it is found that Menispermum-specific CYP80 gene expansion, as well as tissue-specific expression, has driven BIA diversity in Menispermaceae as compared to other Ranunculales species. This study sheds light on WGD occurrences in early-diverging eudicots and the evolution of diverse BIA biosynthesis.
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Bencilisoquinolinas , Sistema Enzimático del Citocromo P-450 , Menispermaceae , Bencilisoquinolinas/metabolismo , Bencilisoquinolinas/química , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Menispermaceae/genética , Menispermaceae/metabolismo , Menispermaceae/química , Alcaloides/metabolismo , Filogenia , Evolución Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Cepharanthine is a secondary metabolite isolated from Stephania. It has been reported that it has anti-conronaviruses activities including severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Here, we assemble three Stephania genomes (S. japonica, S. yunnanensis, and S. cepharantha), propose the cepharanthine biosynthetic pathway, and assess the antiviral potential of compounds involved in the pathway. Among the three genomes, S. japonica has a near telomere-to-telomere assembly with one remaining gap, and S. cepharantha and S. yunnanensis have chromosome-level assemblies. Following by biosynthetic gene mining and metabolomics analysis, we identify seven cepharanthine analogs that have broad-spectrum anti-coronavirus activities, including SARS-CoV-2, Guangxi pangolin-CoV (GX_P2V), swine acute diarrhoea syndrome coronavirus (SADS-CoV), and porcine epidemic diarrhea virus (PEDV). We also show that two other genera, Nelumbo and Thalictrum, can produce cepharanthine analogs, and thus have the potential for antiviral compound discovery. Results generated from this study could accelerate broad-spectrum anti-coronavirus drug discovery.
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Alphacoronavirus , Benzodioxoles , Bencilisoquinolinas , Stephania , Animales , Porcinos , China/epidemiología , SARS-CoV-2 , Antivirales/farmacologíaRESUMEN
Phage therapy has become a viable antimicrobial treatment as an alternative to antibiotic treatment, with an increase in antibiotic resistance. Phage resistance is a major limitation in the therapeutic application of phages, and the lack of understanding of the dynamic changes between bacteria and phages constrains our response strategies to phage resistance. In this study, we investigated the changing trends of mutual resistance between Stenotrophomonas maltophilia (S. maltophilia) and its lytic phage, BUCT603. Our results revealed that S. maltophilia resisted phage infection through mutations in the cell membrane proteins, while the evolved phage re-infected the resistant strain primarily through mutations in structure-related proteins. Compared with the wild-type strain (SMA118), the evolved phage-resistant strain (R118-2) showed reduced virulence, weakened biofilm formation ability, and reduced resistance to aminoglycosides. In addition, the evolved phage BUCT603B1 in combination with kanamycin could inhibit the development of phage-resistant S. maltophilia in vitro and significantly improve the survival rate of S. maltophilia-infected mice. Altogether, these results suggest that in vitro characterization of bacteria-phage co-evolutionary relationships is a useful research tool to optimize phages for the treatment of drug-resistant bacterial infections.IMPORTANCEPhage therapy is a promising approach to treat infections caused by drug-resistant Stenotrophomonas maltophilia (S. maltophilia). However, the rapid development of phage resistance has hindered the therapeutic application of phages. In vitro evolutionary studies of bacteria-phage co-cultures can elucidate the mechanism of resistance development between phage and its host. In this study, we investigated the resistance trends between S. maltophilia and its phage and found that inhibition of phage adsorption is the primary strategy by which bacteria resist phage infection in vitro, while phages can re-infect bacterial cells by identifying other adsorption receptors. Although the final bacterial mutants were no longer infected by phages, they incurred a fitness cost that resulted in a significant reduction in virulence. In addition, the combination treatment with phage and aminoglycoside antibiotics could prevent the development of phage resistance in S. maltophilia in vitro. These findings contribute to increasing the understanding of the co-evolutionary relationships between phages and S. maltophilia.
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Bacteriófagos , Stenotrophomonas maltophilia , Animales , Ratones , Antibacterianos/farmacología , Bacteriófagos/fisiología , Mutación , Stenotrophomonas maltophilia/efectos de los fármacos , Stenotrophomonas maltophilia/genética , Stenotrophomonas maltophilia/virología , Farmacorresistencia Bacteriana , Evolución BiológicaRESUMEN
BACKGROUND: Enterovirus A71 (EV-A71) is capable of causing hand, foot and mouth disease (HFMD), which may lead to neurological sequelae and even death. As EV-A71 is resistant to environmental changes and mutates easily, there is still a lack of effective treatments or globally available vaccines. AIM OF REVIEW: For more than 50 years since the HFMD epidemic, related drug research has been conducted. Progress in this area can promote the further application of existing potential drugs and develop more efficient and safe antiviral drugs, and provide useful reference for protecting the younger generation and maintaining public health security. KEY SCIENTIFIC CONCEPTS OF REVIEW: At present, researchers have identified hundreds of EV-A71 inhibitors based on screening repurposed drugs, targeted structural design, and rational modification of previously effective drugs as the main development strategies. This review systematically introduces the current potential drugs to inhibit EV-A71 infection, including viral inhibitors targeting key sites such as the viral capsid, RNA-dependent RNA polymerase (RdRp), 2C protein, internal ribosome entry site (IRES), 3C proteinase (3Cpro), and 2A proteinase (2Apro), starting from each stage of the viral life cycle. Meanwhile, the progress of host-targeting antiviral drugs and their development are summarized in terms of regulating host immunity, inhibiting autophagy or apoptosis, and regulating the cellular redox environment. In addition, the current clinical methods for the prevention and treatment of HFMD are summarized and discussed with the aim of providing support and recommendations for the treatment of enterovirus infections including EV-A71.
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Enterovirus Humano A , Infecciones por Enterovirus , Enterovirus , Humanos , Enterovirus Humano A/genética , Infecciones por Enterovirus/tratamiento farmacológico , Desarrollo de Medicamentos , Antivirales/farmacología , Antivirales/uso terapéuticoRESUMEN
Mutation of residue 313 in the viral nucleoprotein from F/L to Y/V (or substitutions to N, H, or Q in the nucleoprotein residue 52 adjacent to residue 313) facilitates IAVs to escape from BTN3A3 restriction on virus replication.
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The emergence of phage-resistant bacterial strains is one of the biggest challenges for phage therapy. However, the emerging phage-resistant bacteria are often accompanied by adaptive trade-offs, which supports a therapeutic strategy called "phage steering". The key to phage steering is to guide the bacterial population toward an evolutionary direction that is favorable for treatment. Thus, it is important to systematically investigate the impacts of phages targeting different bacterial receptors on the fitness of the bacterial population. Herein, we employed 20 different phages to impose strong evolutionary pressure on the host Pseudomonas aeruginosa PAO1 and examined the genetic and phenotypic responses of their phage-resistant mutants. Among these strains with impaired adsorptions, four types of mutations associated with bacterial receptors were identified, namely, lipopolysaccharides (LPSs), type IV pili (T4Ps), outer membrane proteins (OMPs), and exopolysaccharides (EPSs). PAO1, responding to LPS- and EPS-dependent phage infections, mostly showed significant growth impairment and virulence attenuation. Most mutants with T4P-related mutations exhibited a significant decrease in motility and biofilm formation ability, while the mutants with OMP-related mutations required the lowest fitness cost out of the bacterial populations. Apart from fitness costs, PAO1 strains might lose their resistance to antibiotics when counteracting with phages, such as the presence of large-fragment mutants in this study, which may inspire the usage of phage-antibiotic combination strategies. This work provides methods that leverage the merits of phage resistance relative to obtaining therapeutically beneficial outcomes with respect to phage-steering strategies.
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Bacteriófagos , Bacteriófagos/genética , Virulencia , Lipopolisacáridos , Evolución Biológica , Antibacterianos , Pseudomonas aeruginosa/fisiologíaRESUMEN
BACKGROUND: Identification and characterization of novel, faithful and processive DNA polymerases is a driving force in the development of DNA amplification methods. Purification of proteins from natural phages is often time-consuming, cumbersome and low yielding. Escherichia coli is a host bacterium widely used for the production of recombinant proteins, is the cell factory of choice for in vitro studies of phage protein function. RESULTS: We expressed the gene encoding Enterococcus faecium phage IME199 DNA polymerase (IME199 DNAP) in Escherichia coli BL21(DE3), and characterized protein function. IME199 DNAP has 3'-5' exonuclease activity, but does not have 5'-3' exonuclease activity. In addition, IME199 DNAP has dNTP-dependent 5'-3' polymerase activity and can amplify DNA at 15-35 °C and a pH range of 5.5-9.5. The amino acid residues Asp30, Glu32, Asp112 and Asp251 are the 3'-5' exonuclease active sites of IME199 DNAP, while residues Asp596 and Tyr639 are essential for DNA synthesis by IME199 DNAP. More importantly, the IME199 DNAP has strand displacement and processive synthesis capabilities, and can perform rolling circle amplification and multiple displacement amplification with very low error rates (approximately 3.67 × 10-6). CONCLUSIONS: A novel family B DNA polymerase was successfully overproduced in Escherichia coli BL21(DE3). Based on the characterized properties, IME199 DNAP is expected to be developed as a high-fidelity polymerase for DNA amplification at room temperature.
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Bacteriófagos , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Bacteriófagos/genética , Enterococcus/metabolismo , Fosfodiesterasa I , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , ADNRESUMEN
Omicron BF.7 became the predominant SARS-CoV-2 variant in Beijing after the abolishment of Zero-COVID policy in December 2022. The ability of antibodies elicited by BF.7 infection to cross-react with SARS-CoV-2-like viruses is unknown. This study aimed to investigate the cross-reactive neutralizing antibodies against SARS-CoV-2-related pangolin coronavirus GX_P2V in sera from vaccinated and/or SARS-CoV-2-infected individuals. All vaccinated individuals who recovered from Omicron BF.7 breakthrough infections exhibited substantially higher levels of neutralizing antibodies against GX_P2V, compared to other subject groups, with a geometric mean titer (GMT) of 362. Uninfected individuals who received four-mixed-dose vaccines also demonstrated higher levels of neutralizing antibodies (GMT = 44) against GX_P2V than those uninfected individuals who received two- or three-dose vaccines and those unvaccinated convalescents of wild-type SARS-CoV-2. This study highlights the significance of prior vaccinations with wild-type SARS-CoV-2 vaccines in generating potent cross-protective immunity against future spillovers of SARS-CoV-2-like viruses.
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COVID-19 , SARS-CoV-2 , Humanos , Animales , SARS-CoV-2/genética , Anticuerpos Neutralizantes , Pangolines , Infección Irruptiva , Vacunas contra la COVID-19 , Anticuerpos AntiviralesRESUMEN
Phage contamination has become a major concern for industrial bacteria, such as Escherichia coli BL21(DE3), used in fermentation processes. Herein, we report a CRISPR/Cas9 defense system-based strategy to precisely prey and degrade phage DNA to decontaminate target phages. First, we isolated a novel phage from fermentation substrates with BL21(DE3) as the host, named TR1. It showed a typical podovirus morphology with a head diameter of 51.46 ± 2.04 nm and a tail length of 9.31 ± 2.77 nm. The burst size of phage TR1 was 151 PFU/cell, suggesting its strong fecundity in the fermentation system. Additionally, whole-genome sequencing revealed that phage TR1 has a DNA genome of 44,099 bp in length with a 43.8% GC content, encoding a total of 68 open reading frames. Comparative genomics and phylogenetic analysis designated this phage to be a new species of the genus Christensenvirus. To counteract phage TR1, we employed the CRISPR/Cas9 system-based strategy and constructed two phage-resistant E. coli strains, BL21-C and BL21-T, based on conserved genes. Both EOP assays and growth curves indicated strong phage resistance of the recombinant strains, without affecting cell growth. Therefore, this study aimed to provide a resilient strategy to respond to ever-changing phages and ongoing phage-host arm race in industrial fermentation environments by the personalized design of spacers in the recombinant CRISPR/Cas system-containing plasmid. More importantly, our research sparks the use of phage defense mechanism to prevent phage contamination in extensive biotechnological applications.
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Multiple LDLR class A (LA) repeats around LA3 promote synergistic binding to Semliki Forest virus (SFV) E1-DIII near the 2-fold and 5-fold symmetry axes. Meanwhile, the multiple consecutive LAs concatemer shows approximately 1000 times higher binding affinity than that of LA3s, which can help to effectively and synergistically bind with E1-DIII of viral envelope protein.
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Cepharanthine, a natural bisbenzylisoquinoline (BBIQ) alkaloid isolated from the plant Stephania Cephalantha Hayata, is the only bisbenzylisoquinoline alkaloid approved for human use and has been used in the clinic for more than 70 years. Cepharanthine has a variety of medicinal properties, including signaling pathway inhibitory activities, immunomodulatory activities, and antiviral activities. Recently, cepharanthine has been confirmed to greatly inhibit SARS-CoV-2 infection. Therefore, we aimed to describe the pharmacological properties and mechanisms of cepharanthine, mainly including antitumor, anti-inflammatory, anti-pathogen activities, inhibition of bone resorption, treatment of alopecia, treatment of snake bite, and other activities. At the same time, we analyzed and summarized the potential antiviral mechanism of cepharanthine and concluded that one of the most important anti-viral mechanisms of cepharanthine may be the stability of plasma membrane fluidity. Additionally, we explained its safety and bioavailability, which provides evidence for cepharanthine as a potential drug for the treatment of a variety of diseases. Finally, we further discuss the potential new clinical applications of cepharanthine and provide direction for its future development.
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Alcaloides , Bencilisoquinolinas , COVID-19 , Humanos , SARS-CoV-2 , Bencilisoquinolinas/farmacología , Alcaloides/farmacología , Antivirales/farmacología , Antivirales/uso terapéuticoRESUMEN
Severe infections caused by multidrug-resistant Klebsiella pneumoniae (K. pneumoniae) highlight the need for new therapeutics with activity against this pathogen. Phage therapy is an alternative treatment approach for multidrug-resistant K. pneumoniae infections. Here, we report a novel bacteriophage (phage) BUCT631 that can specifically lyse capsule-type K1 K. pneumoniae. Physiological characterization revealed that phage BUCT631 could rapidly adsorb to the surface of K. pneumoniae and form an obvious halo ring, and it had relatively favorable thermal stability (4-50 â°C) and pH tolerance (pH â= â4-12). In addition, the optimal multiplicity of infection (MOI) of phage BUCT631 was 0.01, and the burst size was approximately 303 âPFU/cell. Genomic analysis showed that phage BUCT631 has double-stranded DNA (total length of 44,812 bp) with a G â+ âC content of 54.1%, and the genome contains 57 open reading frames (ORFs) and no virulence or antibiotic resistance related genes. Based on phylogenetic analysis, phage BUCT631 could be assigned to a new species in the genus Drulisvirus of the subfamily Slopekvirinae. In addition, phage BUCT631 could quickly inhibit the growth of K. pneumoniae within 2 âh in vitro and significantly elevated the survival rate of K. pneumoniae infected Galleria mellonella larvae from 10% to 90% in vivo. These studies suggest that phage BUCT631 has promising potential for development as a safe alternative for control and treatment of multidrug-resistant K. pneumoniae infection.
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Bacteriófagos , Infecciones por Klebsiella , Mariposas Nocturnas , Animales , Bacteriófagos/genética , Klebsiella pneumoniae/genética , Larva , Filogenia , Infecciones por Klebsiella/terapiaRESUMEN
Artemisinin (ART) and its derivatives have great therapeutical utility as antimalarials and can be repurposed for other indications, such as viral infections, autoimmune diseases, and cancer. This review presents a comprehensive overview of the therapeutic effects of ART-based drugs, beyond their antimalarial effects. This review also summarizes the information on their repurposing in other pathologies, with the hope that it will guide the future optimization of the use of ART-based drugs and of the treatment strategies for the listed diseases. By reviewing related literature, ART extraction and structure as well as the synthesis and structure of its derivatives are presented. Subsequently, the traditional roles of ART and its derivatives against malaria are reviewed, including antimalarial mechanism and occurrence of antimalarial resistance. Finally, the potential of ART and its derivatives to be repurposed for the treatment of other diseases are summarized. The great repurposing potential of ART and its derivatives may be useful for the control of emerging diseases with corresponding pathologies, and future research should be directed toward the synthesis of more effective derivatives or better combinations.
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Antimaláricos , Artemisininas , Antagonistas del Ácido Fólico , Malaria , Humanos , Antimaláricos/uso terapéutico , Antimaláricos/química , Antimaláricos/farmacología , Reposicionamiento de Medicamentos , Artemisininas/uso terapéutico , Artemisininas/química , Artemisininas/farmacología , Malaria/tratamiento farmacológico , Antagonistas del Ácido Fólico/uso terapéuticoRESUMEN
Rotavirus (RV) causes 200,000 deaths per year and imposes a serious burden to public health and livestock farming worldwide. Currently, rehydration (oral and intravenous) remains the main strategy for the treatment of rotavirus gastroenteritis (RVGE), and no specific drugs are available. This review discusses the viral replication cycle in detail and outlines possible therapeutic approaches including immunotherapy, probiotic-assisted therapy, anti-enteric secretory drugs, Chinese medicine, and natural compounds. We present the latest advances in the field of rotavirus antivirals and highlights the potential use of Chinese medicine and natural compounds as therapeutic agents. This review provides an important reference for rotavirus prevention and treatment.
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Gastroenteritis , Infecciones por Rotavirus , Rotavirus , Humanos , Infecciones por Rotavirus/tratamiento farmacológico , Infecciones por Rotavirus/prevención & control , Gastroenteritis/tratamiento farmacológico , Gastroenteritis/prevención & control , Antivirales/uso terapéutico , HospitalizaciónRESUMEN
Bacteria have developed different mechanisms to defend against phages, such as preventing phages from being adsorbed on the surface of host bacteria; through the superinfection exclusion (Sie) block of phage's nucleic acid injection; by restricting modification (R-M) systems, CRISPR-Cas, aborting infection (Abi) and other defense systems to interfere with the replication of phage genes in the host; through the quorum sensing (QS) enhancement of phage's resistant effect. At the same time, phages have also evolved a variety of counter-defense strategies, such as degrading extracellular polymeric substances (EPS) that mask receptors or recognize new receptors, thereby regaining the ability to adsorb host cells; modifying its own genes to prevent the R-M systems from recognizing phage genes or evolving proteins that can inhibit the R-M complex; through the gene mutation itself, building nucleus-like compartments or evolving anti-CRISPR (Acr) proteins to resist CRISPR-Cas systems; and by producing antirepressors or blocking the combination of autoinducers (AIs) and its receptors to suppress the QS. The arms race between bacteria and phages is conducive to the coevolution between bacteria and phages. This review details bacterial anti-phage strategies and anti-defense strategies of phages and will provide basic theoretical support for phage therapy while deeply understanding the interaction mechanism between bacteria and phages.
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Bacteriófagos , Terapia de Fagos , Bacteriófagos/genética , Bacterias/genética , Sistemas CRISPR-Cas , MutaciónRESUMEN
We report the complete genome sequence of the phage BUCT-3589, infecting multidrug-resistant Klebsiella pneumoniae 3589. It is a new member of the genus Przondovirus in the family Autographiviridae and possesses a double-stranded DNA (dsDNA) genome of 40,757 bp with 53.13% GC content. The genome sequence will support its use as a therapeutic agent.
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Acinetobacter baumannii (A. baumannii) is one of the most common clinical pathogens and a typical multi-drug resistant (MDR) bacterium. With the increase of drug-resistant A. baumannii infections, it is urgent to find some new treatment strategies, such as phage therapy. In this paper, we described the different drug resistances of A. baumannii and some basic properties of A. baumannii phages, analyzed the interaction between phages and their hosts, and focused on A. baumannii phage therapies. Finally, we discussed the chance and challenge of phage therapy. This paper aims to provide a more comprehensive understanding of A. baumannii phages and theoretical support for the clinical application of A. baumannii phages.