Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has caused a devastating pandemic worldwide.Vaccines and antiviral drugs are the most promising candidates for combating this global epidemic,and scientists a...Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has caused a devastating pandemic worldwide.Vaccines and antiviral drugs are the most promising candidates for combating this global epidemic,and scientists all over the world have made great efforts to this end.However,manipulation of the SARS-CoV-2 should be performed in the biosafety level3 laboratory.This makes experiments complicated and time-consuming.Therefore,a safer system for working with this virus is urgently needed.Here,we report the construction of plasmid-based,non-infectious SARS-CoV-2 replicons with turbo-green fluorescent protein and/or firefly luciferase reporters by reverse genetics using transformation-associated recombination cloning in Saccharomyces cerevisiae.Replication of these replicons was achieved simply by direct transfection of cells with the replicon plasmids as evident by the expression of reporter genes.Using SARS-CoV-2 replicons,the inhibitory effects of E64-D and remdesivir on SARS-CoV-2 replication were confirmed,and the halfmaximal effective concentration(EC50)value of remdesivir and E64-D was estimated by different quantification methods respectively,indicating that these SARS-CoV-2 replicons are useful tools for antiviral drug evaluation.展开更多
Several variants of concern(VOCs)have emerged since the WIV04 strain of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)was first isolated in January 2020.Due to mutations in the spike(S)protein,these VOCs ...Several variants of concern(VOCs)have emerged since the WIV04 strain of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)was first isolated in January 2020.Due to mutations in the spike(S)protein,these VOCs have evolved to enhance viral infectivity and immune evasion.However,whether mutations of the other viral proteins lead to altered viral propagation and drug resistance remains obscure.The replicon is a noninfectious viral surrogate capable of recapitulating certain steps of the viral life cycle.Although several SARS-CoV-2 replicons have been developed,none of them were derived from emerging VOCs and could only recapitulate viral genome replication and subgenomic RNA(sgRNA)transcription.In this study,SARS-CoV-2 replicons derived from the WIV04 strain and two VOCs(the Beta and Delta variants)were prepared by removing the S gene from their genomes,while other structural genes remained untouched.These replicons not only recapitulate viral genome replication and sgRNA transcription but also support the assembly and release of viral-like particles,as manifested by electron microscopic assays.Thus,the S-deletion replicon could recapitulate virtually all the post-entry steps of the viral life cycle and provides a versatile tool for measuring viral intracellular propagation and screening novel antiviral drugs,including inhibitors of virion assembly and release.Through the quantification of replicon RNA released into the supernatant,we demonstrate that viral intracellular propagation and drug response to remdesivir have not yet substantially changed during the evolution of SARS-CoV-2 from the WIV04 strain to the Beta and Delta VOCs.展开更多
The 2016 Lasker-DeBakey Clinical Medical Research Award was given to three scientists working on different stages of the translational sciences on bringing a high efficacious therapy against hepatitis C virus （HCV） ...The 2016 Lasker-DeBakey Clinical Medical Research Award was given to three scientists working on different stages of the translational sciences on bringing a high efficacious therapy against hepatitis C virus （HCV） infection to a reality. An effective treatment of HCV chronic infection was developed, by a team led by Michael Sofia, using a prodrug approach and the drug PSI-7977 or Sofosbuvir was approved in 2013 less than 28 years after the initial discovery of HCV.展开更多
Rift Valley fever virus(RVFV)is a high-containment pathogen that causes severe diseases in humans,with no approved therapeutics available.Its classification as a biosafety level 3(BSL-3)agent has limited research and ...Rift Valley fever virus(RVFV)is a high-containment pathogen that causes severe diseases in humans,with no approved therapeutics available.Its classification as a biosafety level 3(BSL-3)agent has limited research and therapeutic development due to safety concerns.In this study,we developed a stable replicon cell line maintaining the replication of L and S genomic segments of RVFV.Single-cycle viral replicon particles(VRPs)could be efficiently packaged through trans-complementation of glycoproteins from different strains,recapitulating authentic viral entry and replication while minimizing biosafety risks.Using this system,we conducted high-throughput screening of a small-molecule compound library and identified CNX-1351 as an antiviral agent for multiple RNA viruses.Mechanistic studies revealed that CNX-1351 inhibits viral replication,potentially by targeting the PI3K-Akt signaling pathway.This single-cycle VRP system provides a valuable tool for studying RVFV biology,host interactions,antiviral and vaccine development under reduced biosafety constraints.展开更多
The recent emergence of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)caused serious harm to human health and struck a blow to global economic development.Research on SARS-CoV-2 has greatly benefited from...The recent emergence of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)caused serious harm to human health and struck a blow to global economic development.Research on SARS-CoV-2 has greatly benefited from the use of reverse genetics systems,which have been established to artificially manipulate the viral genome,generating recombinant and reporter infectious viruses or biosafety level 2(BSL-2)-adapted non-infectious replicons with desired modifications.These tools have been instrumental in studying the molecular biological characteristics of the virus,investigating antiviral therapeutics,and facilitating the development of attenuated vaccine candidates.Here,we review the construction strategies,development,and applications of reverse genetics systems for SARS-CoV-2,which may be applied to other CoVs as well.展开更多
SARS-CoV-2 causes the pandemic of COVID-19 and no effective drugs for this disease are available thus far.Due to the high infectivity and pathogenicity of this virus,all studies on the live virus are strictly confined...SARS-CoV-2 causes the pandemic of COVID-19 and no effective drugs for this disease are available thus far.Due to the high infectivity and pathogenicity of this virus,all studies on the live virus are strictly confined in the biosafety level 3(BSL3)laboratory but this would hinder the basic research and antiviral drug development of SARS-CoV-2 because the BSL3 facility is not commonly available and the work in the containment is costly and laborious.In this study,we constructed a reverse genetics system of SARS-CoV-2 by assembling the viral cDNA in a bacterial artificial chromosome(BAC)vector with deletion of the spike(S)gene.Transfection of the cDNA into cells results in the production of an RNA replicon that keeps the capability of genome or subgenome replication but is deficient in virion assembly and infection due to the absence of S protein.Therefore,such a replicon system is not infectious and can be used in ordinary biological laboratories.We confirmed the efficient replication of the replicon by demonstrating the expression of the subgenomic RNAs which have similar profiles to the wild-type virus.By mutational analysis of nsp12 and nsp14,we showed that the RNA polymerase,exonuclease,and cap N7 methyltransferase play essential roles in genome replication and sgRNA production.We also created a SARS-CoV-2 replicon carrying a luciferase reporter gene and this system was validated by the inhibition assays with known anti-SARS-CoV-2 inhibitors.Thus,such a one-plasmid system is biosafe and convenient to use,which will benefit both fundamental research and development of antiviral drugs.展开更多
AIM:To elucidate the effect of antioxidants,resveratrol (RVT)and astaxanthin(AXN),on hepatitis C virus(HCV) replication. METHODS:We investigated the effect of recent popular antioxidant supplements on replication of t...AIM:To elucidate the effect of antioxidants,resveratrol (RVT)and astaxanthin(AXN),on hepatitis C virus(HCV) replication. METHODS:We investigated the effect of recent popular antioxidant supplements on replication of the HCV replicon system OR6.RVT is a strong antioxidant and a kind of polyphenol that inhibits replication of various viruses.AXN is also a strong antioxidant.The replication of HCV RNA was assessed by the luciferase reporter assay.An additive effect of antioxidants on antiviral effects of interferon(IFN)and ribavirin(RBV) was investigated.RESULTS:This is the first report to investigate the effect of RVT and AXN on HCV replication.In contrast to other reported viruses,RVT significantly enhanced HCV RNA replication.Vitamin E also enhanced HCV RNA replication as reported previously,although AXN didnot affect replication.IFN and RBV significantly reduced HCV RNA replication,but these effects were dose-dependently hampered and attenuated by the addition of RVT.AXN didnot affect antiviral effects of IFN or RBV. CONCLUSION:These results suggested that RVT is not suitable as an antioxidant therapy for chronic hepatitis C.展开更多
Objective： The combination of interferon （IFN） and ribavirin （RBV） is the standard therapy for hepatitis C virus （HCV） infection. HCV genotype 2a has proved more amenable to the therapy, but its efficacy is yet...Objective： The combination of interferon （IFN） and ribavirin （RBV） is the standard therapy for hepatitis C virus （HCV） infection. HCV genotype 2a has proved more amenable to the therapy, but its efficacy is yet fimited. This study aimed to investigate the mechanism of the poor response in a case ofHCV genotype 2a infection. Methods： We analyzed dynamic change of HCV RNA from a patient, infected with HCV genotype 2a, showing a poor virological response to 1FN/RBV as judged 12 weeks after initiation of the therapy by HCV clone sequencing. Then we constructed subgenomic Japanese fulminant hepatitis-1 （JFH1） replicon and different chimeric replicons with humanized Gaussia luciferase gene. The chimeric replicons were derived from subgenomic JFH1 replicon, in which the NS5A region was replaced by the patient＇s sequence from the pre/post- treatment, and the chimeric replicons＇ susceptibility to IFN were evaluated by relative Gausia Luciferase activity. Results： The pretreatment HCV sequences appeared almost uniform, and the quasispecies variation was further more simplified after 12 weeks of therapy. Besides, the quasispecies variation seemed to be more diversified in the NS5A, relatively, a region crucial for IFN response, and each of chimeric replicons exhibited distinct response to IFN. Conclusions： During the course of the chronic infection, HCV population seems to be adapted to the patient＇s immunological system, and further to be selected by combination of 1FN/RBV therapy, indicating quasispecies may completely eliminated by addition of other drugs with targets different from those of IFN. In addition, each different response of chimeric replicon to IFN is most likely related to amino acid changes in or near the IFN-sensitivity determining region （ISDR） of NSSA during chronic infection and IFN/RBV therapy.展开更多
Coxsackievirus A10(CVA10)is one of the major etiological agents of hand,foot,and mouth disease.There are no vaccine and antiviral drugs for controlling CVA10 infection.Reverse genetic tools for CVA10 will benefit its ...Coxsackievirus A10(CVA10)is one of the major etiological agents of hand,foot,and mouth disease.There are no vaccine and antiviral drugs for controlling CVA10 infection.Reverse genetic tools for CVA10 will benefit its mechanistic study and development of vaccines and antivirals.Here,two infectious clones for the prototype and a Myc-tagged CVA10 were constructed.Viable CVA10 viruses were harvested by transfecting the viral m RNA into human rhabdomyosarcoma(RD)cells.Rescued CVA10 was further confirmed by next generation sequencing and characterized experimentally.We also constructed the vectors for CVA10 subgenomic replicon with luciferase reporter and viral capsid with EGFP reporter,respectively.Co-transfection of the viral replicon RNA and capsid expresser in human embryonic kidney 293 T(HEK293 T)cells led to the production of single round infectious particles(SRIPs).Based on CVA10 replicon RNA,SRIPs with either the enterovirus A71(EVA71)capsid or the CVA10 capsid were generated.Infection by EVA71 SRIPs required SCARB2,while CVA10 SRIPs did not.Finally,we showed great improvement of the replicon activity and SRIPs production by insertion of a cis-active hammerhead ribozyme(HHRib)before the 50-untranslated region(UTR).In summary,reverse genetic tools for prototype strain of CVA10,including both the infectious clone and the SRIPs system,were successfully established.These tools will facilitate the basic and translational study of CVA10.展开更多
AIM:To evaluate the effects of fucoidan,a complex sulfated polysaccharide extract from marine seaweed,on hepatitis C virus(HCV) RNA load both in vitro and in vivo.METHODS:HCV-1b replicon-expressing cells were cultured...AIM:To evaluate the effects of fucoidan,a complex sulfated polysaccharide extract from marine seaweed,on hepatitis C virus(HCV) RNA load both in vitro and in vivo.METHODS:HCV-1b replicon-expressing cells were cultured in the presence of fucoidan obtained from Cladosiphon okamuranus Tokida cultivated in Okinawa,Japan,and quantified the level of HCV replication.In an open-label uncontrolled study,15 patients with chronic hepatitis C,and HCV-related cirrhosis and hepatocellular carcinoma were treated with fucoidan(0.83 g/d) for 12 mo.The clinical symptoms,biochemical tests,and HCV RNA levels were assessed before,during,and after treatment.RESULTS:Fucoidan dose-dependently inhibited the expression of HCV replicon.At 8-10 mo of treatment with fucoidan,HCV RNA levels were significantly lower relative to the baseline.The same treatment also tended to lower serum alanine aminotransferase levels,and the latter correlated with HCV RNA levels.However,the improved laboratory tests did not translate into significant clinical improvement.Fucoidan had no serious adverse effects.CONCLUSION:Our findings suggest that fucoidan is safe and useful in the treatment of patients with HCVrelated chronic liver diseases.Further controlled clinical trials are needed to confirm the present findings.展开更多
Subgenomic replicons of hepatitis C virus （HCV） have been widely used for studying HCV replication.Here,we report a new subgenomic replicon based on a strain isolated from a chronically infected patient.The coding s...Subgenomic replicons of hepatitis C virus （HCV） have been widely used for studying HCV replication.Here,we report a new subgenomic replicon based on a strain isolated from a chronically infected patient.The coding sequence of HCV was recovered from a Chinese chronic hepatitis C patient displaying high serum HCV copy numbers.A consensus sequence designated as CCH strain was constructed based on the sequences of five clones and this was classified by sequence alignment as belonging to genotype 2a.The subgenomic replicon of CCH was replication-deficient in cell culture,due to dysfunctions in NS3 and NS5B.Various JFH1/CCH chimeric replicons were constructed,and specific mutations were introduced.The introduction of mutations could partially restore the replication of chimeric replicons.A replication-competent chimeric construct was finally obtained by the introduction of NS3 from JFH1 into the backbone of the CCH strain.展开更多
We developed a Gaussia luciferase (Gluc) reporter replicon of West Nile virus (WNV) and used it to quantify viral translation and RNA replication. The advantage of the Gluc replicon is that Gaussia luciferase is secre...We developed a Gaussia luciferase (Gluc) reporter replicon of West Nile virus (WNV) and used it to quantify viral translation and RNA replication. The advantage of the Gluc replicon is that Gaussia luciferase is secreted into the culture medium from cells transfected with Gluc replicon RNA, and the medium can be assayed directly for luciferase activity. Using a known Flavivirus inhibitor (NITD008), we demonstrated that the Gluc-WNV replicon could be used for antiviral screening. The Gluc-WNV-Rep will be useful for research in antiviral drug development programs, as well as for studying viral replication and pathogenesis of WNV.展开更多
AIM: To construct a tricistronic hepatitis C virus (HCV) replicon with double internal ribosome entry sites (IRESes) of only 22 nucleotides for each, substituting the encephalomyocarditis virus (EMCV) IRESes, which ar...AIM: To construct a tricistronic hepatitis C virus (HCV) replicon with double internal ribosome entry sites (IRESes) of only 22 nucleotides for each, substituting the encephalomyocarditis virus (EMCV) IRESes, which are most often used as the translation initiation element to form HCV replicons.展开更多
Middle East respiratory syndrome coronavirus(MERS-CoV)is the causative agent of a severe respiratory disease with a high mortality of~35%.The lack of approved treatments for MERS-CoV infection underscores the need for...Middle East respiratory syndrome coronavirus(MERS-CoV)is the causative agent of a severe respiratory disease with a high mortality of~35%.The lack of approved treatments for MERS-CoV infection underscores the need for a user-friendly system for rapid drug screening.In this study,we constructed a MERS-CoV replicon containing the Renilla luciferase(Rluc)reporter gene and a stable luciferase replicon-carrying cell line.Using this cell line,we showed that MERS-CoV replication was inhibited by combined application of lopinavir and ritonavir,indicating that this cell line can be used to screen inhibitors of MERS-CoV replication.Importantly,the MERS-replicon cell line can be used for high-throughput screening of antiviral drugs without the need for live virus handling,providing an effective and safe tool for the discovery of antiviral drugs against MERS-CoV.展开更多
AIM: To determine if calnexin(CANX), RAB1 and alphatubulin were involved in the production of hepatitis C virus(HCV) particles by baby hamster kidney-West Nile virus(BHK-WNV) cells. METHODS: Using a si RNA-based appro...AIM: To determine if calnexin(CANX), RAB1 and alphatubulin were involved in the production of hepatitis C virus(HCV) particles by baby hamster kidney-West Nile virus(BHK-WNV) cells. METHODS: Using a si RNA-based approach complemented with immuno-fluorescence confocal microscope and Western blot studies, we examined the roles of CANX, RAB1 and alpha-tubulin in the production of HCV particles by permissive BHK-WNV cells expressing HCV structural proteins or the full-length genome of HCV genotype 1a. Immuno-fluorescence studies in producer cells were performed with monoclonal antibodies against HCV structural proteins, as well as immunoglobulin from the serum of a patient recently cured from an HCV infection of same genotype. The cellular compartment stained by the serum immunoglobulin was also observedin thin section transmission electron microscopy. These findings were compared with the JFH-1 strain/Huh-7.5 cell model.RESULTS: We found that CANX was necessary for the production of HCV particles by BHK-WNV cells. This process involved the recruitment of a subset of HCV proteins, detected by immunoglobulin of an HCV-cured patient, in a compartment of rearranged membranes bypassing the endoplasmic reticulum-Golgi intermediary compartment and surrounded by mitochondria. It also involved the maturation of N-linked glycans on HCV envelope proteins, which was required for assembly and/or secretion of HCV particles. The formation of this specialized compartment required RAB1; upon expression of HCV structural genes, this compartment developed large vesicles with viral particles. RAB1 and alpha-tubulin were required for the release of HCV particles. These cellular factors were also involved in the production of HCVcc in the JFH-1 strain/Huh-7.5 cell system, which involves HCV RNA replication. The secretion of HCV particles by BHK-WNV cells presents similarities with a pathway involving caspase-1; a caspase-1 inhibitor was found to suppress the production of HCV particles from a full-length genome.CONCLUSION: Prior activity of the WNV subgenomic replicon in BHK-21 cells promoted re-wiring of host factors for the assembly and release of infectious HCV in a caspase-1-dependent mechanism.展开更多
A high throughput screen of the Pfizer compound collection was carried out using a hepatitis C virus (HCV) genotype 1b subgenomic replicon cell line. Those confirmed hits that demonstrated broad spectrum activity with...A high throughput screen of the Pfizer compound collection was carried out using a hepatitis C virus (HCV) genotype 1b subgenomic replicon cell line. Those confirmed hits that demonstrated broad spectrum activity without overt cytotoxicity were further evaluated, leading to the identification of a series of pyrrolopyridines with excellent antiviral activity in a fully infectious HCV cell-based assay and pharmacokinetic properties.展开更多
CRISPR-based genome editing technologies continue to drive major advances in life sciences.A major challenge for realizing widespread use of genome editing in plants and agriculture is establishing methods that enable...CRISPR-based genome editing technologies continue to drive major advances in life sciences.A major challenge for realizing widespread use of genome editing in plants and agriculture is establishing methods that enable the rapid,comprehensive,and precise evaluation of editing technologies using transient methods.Here we report a new and rapid genome editing evaluation method using Agrobacterium infiltration techniques to enable broad-spectrum,simplistic,and precise assessments of genome editing efficiencies.We employed an anthocyanin marker to facilitate visual screenings of genome-edited cells for use in adult strawberry fruits as well as tomato fruits,cotton leaves,and sugar beet leaves.Using this method,we demonstrate the ability to quickly measure genome editing efficiencies mediated by SpCas9,LbCas12a,A3A-PBE,ABE8e,and PPE.This new method will allow researchers to rapidly and easily evaluate genome editing tools across a broad spectrum of plant species,further expediting the development of genome-edited agricultural crops.展开更多
ADENO-ASSOCIATED virus (AAV) was a human parvovirus considered as a gene delivery vehiclefor human gene therapy. Recombinant AAV vector (rAAV) could mediate the foreign DNAintegration into chromosome and establish a s...ADENO-ASSOCIATED virus (AAV) was a human parvovirus considered as a gene delivery vehiclefor human gene therapy. Recombinant AAV vector (rAAV) could mediate the foreign DNAintegration into chromosome and establish a stable expression in the infected cells. As a viraltransduction vector, rAAV was superior to the traditional retrovirus vector for its high safety.no pathogenicity, and capacity of infecting postmitosis cells. The current strategy for produc-展开更多
A composite EBV based expression vector, pEBAF, was constructed with the 5′ flanking sequence of human α fetoprotein gene as a promoter. Complexed to galactosylated histone, this vector with a foreign DNA insertion ...A composite EBV based expression vector, pEBAF, was constructed with the 5′ flanking sequence of human α fetoprotein gene as a promoter. Complexed to galactosylated histone, this vector with a foreign DNA insertion could be transferred into hepatic cells via the asialoglycoprotein receptor mediated endocytosis pathway. It was replicated as an episome, and its expression was restricted to the AFP positive hepatoma cells. This new gene delivery method has the following advantages: (i) absence of a potential toxicity is related to viral vectors; (ii) the targeting is specific to AFP positive hepatoma cells; (iii) the introduction of the EBV replicon into this system results in the high level expression and long term maintenance of the transferred gene. This novel gene delivery system has potential applications in gene therapy for the treatment of hepatocellular carcinoma.展开更多
基金supported by the CAMS Innovation Fund for Medical Science(CIFMS 2016-I2M-1-014)the National Key Plan for Scientific Research and Development of China(2016YFD0500300)+1 种基金National Key R&D Program of China(2020YFA0707600)Beijing Municipal Science and Technology Project(Z201100001020005)。
文摘Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has caused a devastating pandemic worldwide.Vaccines and antiviral drugs are the most promising candidates for combating this global epidemic,and scientists all over the world have made great efforts to this end.However,manipulation of the SARS-CoV-2 should be performed in the biosafety level3 laboratory.This makes experiments complicated and time-consuming.Therefore,a safer system for working with this virus is urgently needed.Here,we report the construction of plasmid-based,non-infectious SARS-CoV-2 replicons with turbo-green fluorescent protein and/or firefly luciferase reporters by reverse genetics using transformation-associated recombination cloning in Saccharomyces cerevisiae.Replication of these replicons was achieved simply by direct transfection of cells with the replicon plasmids as evident by the expression of reporter genes.Using SARS-CoV-2 replicons,the inhibitory effects of E64-D and remdesivir on SARS-CoV-2 replication were confirmed,and the halfmaximal effective concentration(EC50)value of remdesivir and E64-D was estimated by different quantification methods respectively,indicating that these SARS-CoV-2 replicons are useful tools for antiviral drug evaluation.
基金supported by grants from the National Key Research and Development Project of China (2020YFC0845900)the China Postdoctoral Science Foundation (2020T130021ZX,2021M693198)
文摘Several variants of concern(VOCs)have emerged since the WIV04 strain of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)was first isolated in January 2020.Due to mutations in the spike(S)protein,these VOCs have evolved to enhance viral infectivity and immune evasion.However,whether mutations of the other viral proteins lead to altered viral propagation and drug resistance remains obscure.The replicon is a noninfectious viral surrogate capable of recapitulating certain steps of the viral life cycle.Although several SARS-CoV-2 replicons have been developed,none of them were derived from emerging VOCs and could only recapitulate viral genome replication and subgenomic RNA(sgRNA)transcription.In this study,SARS-CoV-2 replicons derived from the WIV04 strain and two VOCs(the Beta and Delta variants)were prepared by removing the S gene from their genomes,while other structural genes remained untouched.These replicons not only recapitulate viral genome replication and sgRNA transcription but also support the assembly and release of viral-like particles,as manifested by electron microscopic assays.Thus,the S-deletion replicon could recapitulate virtually all the post-entry steps of the viral life cycle and provides a versatile tool for measuring viral intracellular propagation and screening novel antiviral drugs,including inhibitors of virion assembly and release.Through the quantification of replicon RNA released into the supernatant,we demonstrate that viral intracellular propagation and drug response to remdesivir have not yet substantially changed during the evolution of SARS-CoV-2 from the WIV04 strain to the Beta and Delta VOCs.
文摘The 2016 Lasker-DeBakey Clinical Medical Research Award was given to three scientists working on different stages of the translational sciences on bringing a high efficacious therapy against hepatitis C virus （HCV） infection to a reality. An effective treatment of HCV chronic infection was developed, by a team led by Michael Sofia, using a prodrug approach and the drug PSI-7977 or Sofosbuvir was approved in 2013 less than 28 years after the initial discovery of HCV.
基金Grants from the Program of Shanghai Academic Research Leader(22XD1420600)Shanghai Municipal Science and Technology Major Project(ZD2021CY001)+2 种基金National Natural Science Foundation of China(81974305)Shenzhen Medical Research Fund(SMRF No.B2302029)Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences(2023-PT310-02)supported this work.
文摘Rift Valley fever virus(RVFV)is a high-containment pathogen that causes severe diseases in humans,with no approved therapeutics available.Its classification as a biosafety level 3(BSL-3)agent has limited research and therapeutic development due to safety concerns.In this study,we developed a stable replicon cell line maintaining the replication of L and S genomic segments of RVFV.Single-cycle viral replicon particles(VRPs)could be efficiently packaged through trans-complementation of glycoproteins from different strains,recapitulating authentic viral entry and replication while minimizing biosafety risks.Using this system,we conducted high-throughput screening of a small-molecule compound library and identified CNX-1351 as an antiviral agent for multiple RNA viruses.Mechanistic studies revealed that CNX-1351 inhibits viral replication,potentially by targeting the PI3K-Akt signaling pathway.This single-cycle VRP system provides a valuable tool for studying RVFV biology,host interactions,antiviral and vaccine development under reduced biosafety constraints.
基金the Major Research Plan of the National Natural Science Foundation of China(92269105)the Zhejiang Provincial Natural Science Foundation(LZ22C180002).
文摘The recent emergence of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)caused serious harm to human health and struck a blow to global economic development.Research on SARS-CoV-2 has greatly benefited from the use of reverse genetics systems,which have been established to artificially manipulate the viral genome,generating recombinant and reporter infectious viruses or biosafety level 2(BSL-2)-adapted non-infectious replicons with desired modifications.These tools have been instrumental in studying the molecular biological characteristics of the virus,investigating antiviral therapeutics,and facilitating the development of attenuated vaccine candidates.Here,we review the construction strategies,development,and applications of reverse genetics systems for SARS-CoV-2,which may be applied to other CoVs as well.
基金supported by Grants(the National Natural Science Foundation of China#32041002,#31971161,#31900546 and#81620108020)the Guangdong Science and Technology Department(#2019A1515011332)+1 种基金the Shenzhen Science and Technology Innovation Program(JSGG20200225150431472,JCYJ20190807160615255,JCYJ20190807153203560,and KQTD20180411143323605)supported by the Guangdong Zhujiang Leading Talents Programme and the National Tenthousand Talents Program。
文摘SARS-CoV-2 causes the pandemic of COVID-19 and no effective drugs for this disease are available thus far.Due to the high infectivity and pathogenicity of this virus,all studies on the live virus are strictly confined in the biosafety level 3(BSL3)laboratory but this would hinder the basic research and antiviral drug development of SARS-CoV-2 because the BSL3 facility is not commonly available and the work in the containment is costly and laborious.In this study,we constructed a reverse genetics system of SARS-CoV-2 by assembling the viral cDNA in a bacterial artificial chromosome(BAC)vector with deletion of the spike(S)gene.Transfection of the cDNA into cells results in the production of an RNA replicon that keeps the capability of genome or subgenome replication but is deficient in virion assembly and infection due to the absence of S protein.Therefore,such a replicon system is not infectious and can be used in ordinary biological laboratories.We confirmed the efficient replication of the replicon by demonstrating the expression of the subgenomic RNAs which have similar profiles to the wild-type virus.By mutational analysis of nsp12 and nsp14,we showed that the RNA polymerase,exonuclease,and cap N7 methyltransferase play essential roles in genome replication and sgRNA production.We also created a SARS-CoV-2 replicon carrying a luciferase reporter gene and this system was validated by the inhibition assays with known anti-SARS-CoV-2 inhibitors.Thus,such a one-plasmid system is biosafe and convenient to use,which will benefit both fundamental research and development of antiviral drugs.
基金Supported by The grant from the Japanese Ministry of Educationand Science
文摘AIM:To elucidate the effect of antioxidants,resveratrol (RVT)and astaxanthin(AXN),on hepatitis C virus(HCV) replication. METHODS:We investigated the effect of recent popular antioxidant supplements on replication of the HCV replicon system OR6.RVT is a strong antioxidant and a kind of polyphenol that inhibits replication of various viruses.AXN is also a strong antioxidant.The replication of HCV RNA was assessed by the luciferase reporter assay.An additive effect of antioxidants on antiviral effects of interferon(IFN)and ribavirin(RBV) was investigated.RESULTS:This is the first report to investigate the effect of RVT and AXN on HCV replication.In contrast to other reported viruses,RVT significantly enhanced HCV RNA replication.Vitamin E also enhanced HCV RNA replication as reported previously,although AXN didnot affect replication.IFN and RBV significantly reduced HCV RNA replication,but these effects were dose-dependently hampered and attenuated by the addition of RVT.AXN didnot affect antiviral effects of IFN or RBV. CONCLUSION:These results suggested that RVT is not suitable as an antioxidant therapy for chronic hepatitis C.
文摘Objective： The combination of interferon （IFN） and ribavirin （RBV） is the standard therapy for hepatitis C virus （HCV） infection. HCV genotype 2a has proved more amenable to the therapy, but its efficacy is yet fimited. This study aimed to investigate the mechanism of the poor response in a case ofHCV genotype 2a infection. Methods： We analyzed dynamic change of HCV RNA from a patient, infected with HCV genotype 2a, showing a poor virological response to 1FN/RBV as judged 12 weeks after initiation of the therapy by HCV clone sequencing. Then we constructed subgenomic Japanese fulminant hepatitis-1 （JFH1） replicon and different chimeric replicons with humanized Gaussia luciferase gene. The chimeric replicons were derived from subgenomic JFH1 replicon, in which the NS5A region was replaced by the patient＇s sequence from the pre/post- treatment, and the chimeric replicons＇ susceptibility to IFN were evaluated by relative Gausia Luciferase activity. Results： The pretreatment HCV sequences appeared almost uniform, and the quasispecies variation was further more simplified after 12 weeks of therapy. Besides, the quasispecies variation seemed to be more diversified in the NS5A, relatively, a region crucial for IFN response, and each of chimeric replicons exhibited distinct response to IFN. Conclusions： During the course of the chronic infection, HCV population seems to be adapted to the patient＇s immunological system, and further to be selected by combination of 1FN/RBV therapy, indicating quasispecies may completely eliminated by addition of other drugs with targets different from those of IFN. In addition, each different response of chimeric replicon to IFN is most likely related to amino acid changes in or near the IFN-sensitivity determining region （ISDR） of NSSA during chronic infection and IFN/RBV therapy.
基金supported by the Shanghai Public Health Clinical Center(KY-GW2018-17)Shanghai Municipal Commission of Health and Family Planning(20174Y0099)。
文摘Coxsackievirus A10(CVA10)is one of the major etiological agents of hand,foot,and mouth disease.There are no vaccine and antiviral drugs for controlling CVA10 infection.Reverse genetic tools for CVA10 will benefit its mechanistic study and development of vaccines and antivirals.Here,two infectious clones for the prototype and a Myc-tagged CVA10 were constructed.Viable CVA10 viruses were harvested by transfecting the viral m RNA into human rhabdomyosarcoma(RD)cells.Rescued CVA10 was further confirmed by next generation sequencing and characterized experimentally.We also constructed the vectors for CVA10 subgenomic replicon with luciferase reporter and viral capsid with EGFP reporter,respectively.Co-transfection of the viral replicon RNA and capsid expresser in human embryonic kidney 293 T(HEK293 T)cells led to the production of single round infectious particles(SRIPs).Based on CVA10 replicon RNA,SRIPs with either the enterovirus A71(EVA71)capsid or the CVA10 capsid were generated.Infection by EVA71 SRIPs required SCARB2,while CVA10 SRIPs did not.Finally,we showed great improvement of the replicon activity and SRIPs production by insertion of a cis-active hammerhead ribozyme(HHRib)before the 50-untranslated region(UTR).In summary,reverse genetic tools for prototype strain of CVA10,including both the infectious clone and the SRIPs system,were successfully established.These tools will facilitate the basic and translational study of CVA10.
文摘AIM:To evaluate the effects of fucoidan,a complex sulfated polysaccharide extract from marine seaweed,on hepatitis C virus(HCV) RNA load both in vitro and in vivo.METHODS:HCV-1b replicon-expressing cells were cultured in the presence of fucoidan obtained from Cladosiphon okamuranus Tokida cultivated in Okinawa,Japan,and quantified the level of HCV replication.In an open-label uncontrolled study,15 patients with chronic hepatitis C,and HCV-related cirrhosis and hepatocellular carcinoma were treated with fucoidan(0.83 g/d) for 12 mo.The clinical symptoms,biochemical tests,and HCV RNA levels were assessed before,during,and after treatment.RESULTS:Fucoidan dose-dependently inhibited the expression of HCV replicon.At 8-10 mo of treatment with fucoidan,HCV RNA levels were significantly lower relative to the baseline.The same treatment also tended to lower serum alanine aminotransferase levels,and the latter correlated with HCV RNA levels.However,the improved laboratory tests did not translate into significant clinical improvement.Fucoidan had no serious adverse effects.CONCLUSION:Our findings suggest that fucoidan is safe and useful in the treatment of patients with HCVrelated chronic liver diseases.Further controlled clinical trials are needed to confirm the present findings.
基金supported by grants from the National Basic Research Priorities Program of China(2013CB911101)the National Nature Science Foundation of China(Grant 31200315)
文摘Subgenomic replicons of hepatitis C virus （HCV） have been widely used for studying HCV replication.Here,we report a new subgenomic replicon based on a strain isolated from a chronically infected patient.The coding sequence of HCV was recovered from a Chinese chronic hepatitis C patient displaying high serum HCV copy numbers.A consensus sequence designated as CCH strain was constructed based on the sequences of five clones and this was classified by sequence alignment as belonging to genotype 2a.The subgenomic replicon of CCH was replication-deficient in cell culture,due to dysfunctions in NS3 and NS5B.Various JFH1/CCH chimeric replicons were constructed,and specific mutations were introduced.The introduction of mutations could partially restore the replication of chimeric replicons.A replication-competent chimeric construct was finally obtained by the introduction of NS3 from JFH1 into the backbone of the CCH strain.
基金supported by the National Natural Science Foundation of China (grant No. 31170158 and 31000090)the '100 Talents Project' of Chinese Academy of Sciences, China (grant No. Y002171YC1)
文摘We developed a Gaussia luciferase (Gluc) reporter replicon of West Nile virus (WNV) and used it to quantify viral translation and RNA replication. The advantage of the Gluc replicon is that Gaussia luciferase is secreted into the culture medium from cells transfected with Gluc replicon RNA, and the medium can be assayed directly for luciferase activity. Using a known Flavivirus inhibitor (NITD008), we demonstrated that the Gluc-WNV replicon could be used for antiviral screening. The Gluc-WNV-Rep will be useful for research in antiviral drug development programs, as well as for studying viral replication and pathogenesis of WNV.
基金Supported by Grants from the Study on Prevention and Control of Viral Hepatitis in the Key Program for Science and Technology Development of Hebei Province,No.10276102D
文摘AIM: To construct a tricistronic hepatitis C virus (HCV) replicon with double internal ribosome entry sites (IRESes) of only 22 nucleotides for each, substituting the encephalomyocarditis virus (EMCV) IRESes, which are most often used as the translation initiation element to form HCV replicons.
基金This study was supported by the strategic priority research program of the Chinese academy of sciences(XDB29010101).
文摘Middle East respiratory syndrome coronavirus(MERS-CoV)is the causative agent of a severe respiratory disease with a high mortality of~35%.The lack of approved treatments for MERS-CoV infection underscores the need for a user-friendly system for rapid drug screening.In this study,we constructed a MERS-CoV replicon containing the Renilla luciferase(Rluc)reporter gene and a stable luciferase replicon-carrying cell line.Using this cell line,we showed that MERS-CoV replication was inhibited by combined application of lopinavir and ritonavir,indicating that this cell line can be used to screen inhibitors of MERS-CoV replication.Importantly,the MERS-replicon cell line can be used for high-throughput screening of antiviral drugs without the need for live virus handling,providing an effective and safe tool for the discovery of antiviral drugs against MERS-CoV.
基金Supported by Intramural Program of the National Institutes of Health,National Institute of Allergy and Infectious Diseases(Project No.1 ZIA AI000733-15:Enveloped Virus Glycoprotein/Receptor Interactions)to Edward A Berger,PhD(MSS,LVD,DIR,NIAID)ORISE Senior Fellow award(Award No.1238-1238-03:Department of Energy/Oak Ridge Institute for Science and Education)to Bertrand Saunier,MD,PhD
文摘AIM: To determine if calnexin(CANX), RAB1 and alphatubulin were involved in the production of hepatitis C virus(HCV) particles by baby hamster kidney-West Nile virus(BHK-WNV) cells. METHODS: Using a si RNA-based approach complemented with immuno-fluorescence confocal microscope and Western blot studies, we examined the roles of CANX, RAB1 and alpha-tubulin in the production of HCV particles by permissive BHK-WNV cells expressing HCV structural proteins or the full-length genome of HCV genotype 1a. Immuno-fluorescence studies in producer cells were performed with monoclonal antibodies against HCV structural proteins, as well as immunoglobulin from the serum of a patient recently cured from an HCV infection of same genotype. The cellular compartment stained by the serum immunoglobulin was also observedin thin section transmission electron microscopy. These findings were compared with the JFH-1 strain/Huh-7.5 cell model.RESULTS: We found that CANX was necessary for the production of HCV particles by BHK-WNV cells. This process involved the recruitment of a subset of HCV proteins, detected by immunoglobulin of an HCV-cured patient, in a compartment of rearranged membranes bypassing the endoplasmic reticulum-Golgi intermediary compartment and surrounded by mitochondria. It also involved the maturation of N-linked glycans on HCV envelope proteins, which was required for assembly and/or secretion of HCV particles. The formation of this specialized compartment required RAB1; upon expression of HCV structural genes, this compartment developed large vesicles with viral particles. RAB1 and alpha-tubulin were required for the release of HCV particles. These cellular factors were also involved in the production of HCVcc in the JFH-1 strain/Huh-7.5 cell system, which involves HCV RNA replication. The secretion of HCV particles by BHK-WNV cells presents similarities with a pathway involving caspase-1; a caspase-1 inhibitor was found to suppress the production of HCV particles from a full-length genome.CONCLUSION: Prior activity of the WNV subgenomic replicon in BHK-21 cells promoted re-wiring of host factors for the assembly and release of infectious HCV in a caspase-1-dependent mechanism.
文摘A high throughput screen of the Pfizer compound collection was carried out using a hepatitis C virus (HCV) genotype 1b subgenomic replicon cell line. Those confirmed hits that demonstrated broad spectrum activity without overt cytotoxicity were further evaluated, leading to the identification of a series of pyrrolopyridines with excellent antiviral activity in a fully infectious HCV cell-based assay and pharmacokinetic properties.
基金supported by the National Natural Science Foundation of China(31788103 and 31971370)the National Key Research and Development Program(2022YFF1002802)the Ministry of Agriculture and Rural Affairs of China,and the Strategic Priority Research Program of the Chinese Academy of Sciences(Precision Seed Design and Breeding,XDA24020102).
文摘CRISPR-based genome editing technologies continue to drive major advances in life sciences.A major challenge for realizing widespread use of genome editing in plants and agriculture is establishing methods that enable the rapid,comprehensive,and precise evaluation of editing technologies using transient methods.Here we report a new and rapid genome editing evaluation method using Agrobacterium infiltration techniques to enable broad-spectrum,simplistic,and precise assessments of genome editing efficiencies.We employed an anthocyanin marker to facilitate visual screenings of genome-edited cells for use in adult strawberry fruits as well as tomato fruits,cotton leaves,and sugar beet leaves.Using this method,we demonstrate the ability to quickly measure genome editing efficiencies mediated by SpCas9,LbCas12a,A3A-PBE,ABE8e,and PPE.This new method will allow researchers to rapidly and easily evaluate genome editing tools across a broad spectrum of plant species,further expediting the development of genome-edited agricultural crops.
文摘ADENO-ASSOCIATED virus (AAV) was a human parvovirus considered as a gene delivery vehiclefor human gene therapy. Recombinant AAV vector (rAAV) could mediate the foreign DNAintegration into chromosome and establish a stable expression in the infected cells. As a viraltransduction vector, rAAV was superior to the traditional retrovirus vector for its high safety.no pathogenicity, and capacity of infecting postmitosis cells. The current strategy for produc-
文摘A composite EBV based expression vector, pEBAF, was constructed with the 5′ flanking sequence of human α fetoprotein gene as a promoter. Complexed to galactosylated histone, this vector with a foreign DNA insertion could be transferred into hepatic cells via the asialoglycoprotein receptor mediated endocytosis pathway. It was replicated as an episome, and its expression was restricted to the AFP positive hepatoma cells. This new gene delivery method has the following advantages: (i) absence of a potential toxicity is related to viral vectors; (ii) the targeting is specific to AFP positive hepatoma cells; (iii) the introduction of the EBV replicon into this system results in the high level expression and long term maintenance of the transferred gene. This novel gene delivery system has potential applications in gene therapy for the treatment of hepatocellular carcinoma.
