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Generation of Recombinant Equine Influenza Vaccine Candidate RgH3N1 Virus by reverse genetics

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Agricultural Sciences in China 2005年第4期4卷 317-320页

作者： ZHANGYun LIUMing YUKang-zhen WebsterRobert NationalKeyLaboratoryofVeterinaryBiotechnology HarbinVeterinaryResearchInstituteChineseAcademyofAgriculturalSciencesHarbin150001P.R.China NationalKeyLaboratoryofVeterinaryBiotechnology HarbinVeterinaryResearchInstituteChineseAcademyofAgriculturalSciencesHarbin150001P.R.China VirologyDivisionoflnfectiousDiseaseDepartment St.JudeChildren＇sResearchHospitalMemphis38105USA

The antigenic variation of influenza A virus hemagglutinin (HA) glycoproteins requires frequent changes in vaccine formulation. The new strategy of creating influenza seed strains for vaccine production is to generate 7 + 1 reassortants that contain seven genes from a high-yield virus A/Puerto Rico/8/34[A/PR/8/34](H1N1) and the HA gene from the circulating strains. By using this DNA-based cotransfection technique, we generated 7 + 1 reassortants rgH3N1 which had the antigenic determinants of influenza virus A/Songbird/HongKong/102/00[SB/HK/01](H3N8) and 7 other genes from A/PR/ 8/34. The hemagglutinin of A/Songbird/HongKong/102/00 is 96.3% homologous to that of A/Equine/Jilin/98[Eq/Jl/89] (H3N8). The resulting virus rgH3N1 grows to high HA titers in chicken embryonated eggs, allowing vaccine preparation in unconcentrated allantoic fluid. The rgH3N1 is stable after multiple passages in embryonated eggs. The reassortant rgH3N1 virus could be used as vaccine candidate to reduce the reemergence of equine influenza outbreaks.

关键词： Influenza A virus reverse genetics In vitro properties Stability of reassortant virus

Identification of novel small-molecule inhibitors of SARS-CoV-2 by chemical genetics

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Acta Pharmaceutica Sinica B 2024年第9期14卷 4028-4044页

作者： Chris Chun-Yiu Chan Qian Guo Jasper Fuk-Woo Chan Kaiming Tang Jian-Piao Cai Kenn Ka-Heng Chik Yixin Huang Mei Dai Bo Qin Chon Phin Ong Allen Wing-Ho Chu Wan-Mui Chan Jonathan Daniel Ip Lei Wen Jessica Oi-Ling Tsang Tong-Yun Wang Yubin Xie Zhenzhi Qin Jianli Cao Zi-Wei Ye Hin Chu Kelvin Kai-Wang To Xing-Yi Ge Tao Ni Dong-Yan Jin Sheng Cui Kwok-Yung Yuen Shuofeng Yuan State Key Laboratory of Emerging Infectious Diseases Carol Yu Centre for InfectionDepartment of MicrobiologySchool of Clinical MedicineLi Ka Shing Faculty of Medicinethe University of Hong KongPokfulamHong Kong SAR 999077China Department of Infectious Diseases and Microbiology the University of Hong Kong-Shenzhen HospitalShenzhen 518000China Centre for Virology Vaccinology and TherapeuticsHong Kong Science and Technology ParkHong Kong SAR 999077China Academician Workstation of Hainan Province Hainan Medical University-the University of Hong Kong Joint Laboratory of Tropical Infectious DiseasesHaikou 571100China School of Biomedical Sciences Li Ka Shing Faculty of Medicinethe University of Hong KongPokfulamHong Kong SAR 999077China NHC Key Laboratory of Systems Biology of Pathogens Institute of Pathogen Biology Chinese Academy of Medical Sciences&Peking Union Medical CollegeBeijing 100730China College of Biology Hunan Provincial Key Laboratory of Medical VirologyHunan UniversityChangsha 410082China

There are only eight approved small molecule antiviral drugs for treating *** them,four are nucleotide analogues(remdesivir,JT001,molnupiravir,and azvudine),while the other four are protease inhibitors(nirmatrelvir,ensitrelvir,leritrelvir,and simnotrelvir-ritonavir).Antiviral resistance,unfavourable drug‒drug interaction,and toxicity have been reported in previous *** there is a dearth of new treatment options for *** this work,a three-tier cell-based screening was employed to identify novel compounds with anti-SARS-CoV-2 *** compound,designated 172,demonstrated broad-spectrum antiviral activity against multiple human pathogenic coronaviruses and different SARS-CoV-2 variants of *** studies validated by reverse genetics showed that compound 172 inhibits the 3-chymotrypsin-like protease(3CLpro)by binding to an allosteric site and reduces 3CLpro dimerization.A drug synergistic checkerboard assay demonstrated that compound 172 can achieve drug synergy with nirmatrelvir in *** vivo studies confirmed the antiviral activity of compound 172 in both Golden Syrian Hamsters and K18 humanized ACE2 ***,this study identified an alternative druggable site on the SARS-CoV-23CLpro,proposed a potential combination therapy with nirmatrelvir to reduce the risk of antiviral resistance and shed light on the development of allosteric protease inhibitors for treating a range of coronavirus diseases.

关键词： SARS-CoV-2 High throughput screening Broad-spectrum antiviral treatment 3CLpro inhibitor Allosteric-site inhibitor Animal models Chemical genetics reverse genetics

Decoupling gene functions from knockout effects by evolutionary analyses

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National Science Review 2020年第7期7卷 1169-1180页

作者： Li Liu Mengdi Liu Di Zhang Shanjun Deng Piaopiao Chen Jing Yang Yunhan Xie Xionglei He State Key Laboratory of Biocontrol School of Life Sciences Sun Yat-sen University

Genic functions have long been confounded by pleiotropic mutational effects. To understand such genetic effects, we examine HAP4, a well-studied transcription factor in Saccharomyces cerevisiae that functions by forming a tetramer with HAP2, HAP3 and HAP5. Deletion of HAP4 results in highly pleiotropic gene expression responses, some of which are clustered in related cellular processes(clustered effects) while most are distributed randomly across diverse cellular processes(distributed effects). Strikingly, the distributed effects that account for much of HAP4 pleiotropy tend to be non-heritable in a population,suggesting they have few evolutionary consequences. Indeed, these effects are poorly conserved in closely related yeasts. We further show substantial overlaps of clustered effects, but not distributed effects, among the four genes encoding the HAP2/3/4/5 tetramer. This pattern holds for other biochemically characterized yeast protein complexes or metabolic pathways. Examination of a set of cell morphological traits of the deletion lines yields consistent results. Hence, only some deletion effects of a gene support related biochemical understandings with the rest being often pleiotropic and evolutionarily decoupled from the gene's normal functions. This study suggests a new framework for reverse genetic analysis.

关键词： pleiotropy mutational effects heritability reverse genetics gene function

Synthesis of Reassortant Infectious Bursal Disease Virus in Chickens Injected Directly with Infectious Clones from Different Virus Strains

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Acta Biochimica et Biophysica Sinica 2005年第3期37卷 192-198页

作者： Long LI Yao-Wei HUANG Lian-Sheng WANG Wang-Jun WAN Lian YU Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine,Institute of Preventive Veterinary Medicine.Zhejiang University.Hangzhou 310029,China Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine Institute of Preventive Veterinary Medicine Zhejiang UniversityHangzhou 310029 China

The infectious bursal disease virus（IBDV）,a member of the Birnaviridae family,containinga bisegmented double-stranded RNA genome,encodes four structural viral proteins,VP1,VP2,VP3,andVP4,as well as a non-structural protein,VP5,In the present paper,the segment A from two IBDV strains,field isolate ZJ2000 and attenuated strain HZ2,were inserted into one NaeI site by site-directed silent mu-tagenesis and subcloned into the eukaryotic expression plasmid pCI under the control of the human cy-tomegalovirus（hCMV）immediate early enhancer and promoter to construct the recombinant plasmids pCI-AKZJ2000and pCI-AKHZ2,*** of the two recombinants was combined with another recombinant pCIplasroid containing the marked segment B of strain HZ2（pCI-mB）,and injected intramuscularly into non-immunized *** chimeric IBDV strains were recovered from the *** out of eightchickens in each of two groups showed the bursal histopathological *** reassortant virus derivedfrom pCI-AKZJ2000/pCI-mB can infect chicken embryos and shows relatively low *** havedeveloped a novel virus reverse genetic approach for the study of *** results also form the basis forinvestigating the role of VP1 in viral replication and pathogenecity.

关键词： infectious bursal disease infectious bursal disease virus reverse genetics reassortant virus animal model

The Establishment of Infectious Clone and Single Round Infectious Particles for Coxsackievirus A10

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Virologica Sinica 2020年第4期35卷 426-435页

作者： Min Wang Jingjing Yan Liuyao Zhu Meng Wang Lizhen Liu Rui Yu Ming Chen Jingna Xun Yuling Zhang Zhigang Yi Shuye Zhang Shanghai Public Health Clinical Center and Institute of Biomedical Sciences Fudan UniversityShanghai 201508China

Coxsackievirus A10(CVA10)is one of the major etiological agents of hand,foot,and mouth *** are no vaccine and antiviral drugs for controlling CVA10 *** genetic tools for CVA10 will benefit its mechanistic study and development of vaccines and ***,two infectious clones for the prototype and a Myc-tagged CVA10 were *** CVA10 viruses were harvested by transfecting the viral m RNA into human rhabdomyosarcoma(RD)*** CVA10 was further confirmed by next generation sequencing and characterized *** also constructed the vectors for CVA10 subgenomic replicon with luciferase reporter and viral capsid with EGFP reporter,***-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 *** by EVA71 SRIPs required SCARB2,while CVA10 SRIPs did ***,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 *** tools will facilitate the basic and translational study of CVA10.

关键词： Coxsackievirus A10(CVA10) reverse genetics Enterovirus Single round infectious particles(SRIPs) Replicon

Efficient and Specific Modifications of the Drosophila Genome by Means of an Easy TALEN Strategy

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Journal of genetics and Genomics 2012年第5期39卷 209-215页

作者： Jiyong Liu Changqing Li Zhongsheng Yu Peng Huang Honggang Wu Chuanxian Wei Nannan Zhu Yan Shen Yixu Chen Bo Zhang Wu-Min Deng Renjie Jiao State Key Laboratory of Brain and Cognitive Science Institute of Biophysics The Chinese Academy of Sciences Datun Road 15 Beijing 100101 China Key Laboratory of Cell Proliferation and Differentiation of Ministry of Education College of Life Sciences Peking University Beijing 100871 China Department of Biological Science Florida State University Tallahassee Florida 32304-4295 USA

Technology development has always been one of the forces driving breakthroughs in biomedical research. Since the time of Thomas Morgan, Drosophilists have, step by step, developed powerful genetic tools for manipulating and functionally dissecting the Drosophila genome, but room for improving these technologies and developing new techniques is still large, especially today as biologists start to study systematically the functional genomics of different model organisms, including humans, in a high-throughput manner. Here, we report, for the first time in Drosophila, a rapid, easy, and highly specific method for modifying the Drosophila genome at a very high efficiency by means of an improved transcription activator-like effector nuclease （TALEN） strategy. We took advantage of the very recently developed ＂unit assembly＂ strategy to assemble two pairs of specific TALENs designed to modify the yellow gene （on the sex chromosome） and a novel autosomal gene. The mRNAs of TALENs were subsequently injected into Drosophila embryos. From 31.2% of the injected Fo fertile flies, we detected inheritable modification involving the yellow gene. The entire process from construction of specific TALENs to detection of inheritable modifications can be accomplished within one month. The potential applications of this TALEN-mediated genome modification method in Drosophila are discussed.

关键词： TALEN Drosophila Genomic modification Unit assembly reverse genetics

Phytochrome-regulated Gene Expression

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Journal of Integrative Plant Biology 2007年第1期49卷 11-20页

作者： Peter H. Quail Department of Plant and Microbial Biology University of Cafifomia Berkeley CA 94720 USA U.S. Department of Agriculture/Agriculture Research Service-Plant Gene Expression Center 800 Buchanan Street Albany CA 94710 LISA

Identification of all genes involved in the phytochrome （phy）-medieted responses of plants to their light environment is an important goal in providing an overall understanding of light-regulated growth end development. This article highlights end integrates the central findings of two recent comprehensive studies in Arabidopsis that have identified the genome-wide set of phy-reguleted genes that respond rapidly to red-light signals upon first exposure of dark-grown seedlings, and have tested the functional relevance to normal seedling photomorphogenesis of an Initial subset of these genes. The data：（a） reveal considerable complexity in the channeling of the light signals through the different phy-femily members （phyA to phyE） to responsive genes; （b） identify a diversity of transcription-factor-encoding genes as major early, if not primary, targets of phy signaling, end, therefore, as potentially important regulators in the transcriptional-network hierarchy; and （c） identify auxin-related genes as the dominant class among rapidly-regulated, hormone-related genes. However, reverse-genetic functional profiling of a selected subset of these genes reveals that only a limited fraction are necessary for optimal phy-induced seedling deetioletion.

关键词： expression profiling phytochrome signaling transcriptional networks microarrays reverse genetics functional profiling transcription factors auxin-related genes

Now and future of mouse mutagenesis for human disease models

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Journal of genetics and Genomics 2010年第9期37卷 559-572页

作者： Yoichi Gondo Mutagenesis and Genomics Team RIKEN Bio Resource Center

One of the major objectives of the Human Genome Project is to understand the biological function of the gene and genome as well as to develop clinical applications for human diseases. For this purpose, the experimental validations and preclinical trails by using animal models are indispensable. The mouse （Mus musculus） is one of the best animal models because genetics is well established in the mouse and embryonic manipulation technologies are also well developed. Large-scale mouse mutagenesis projects have been conducted to develop various mouse models since 1997. Originally, the phenotype-driven mutagenesis with N-ethyl-N-nitrosourea （ENU） has been the major efforts internationally then knockout/conditional mouse projects and gene-driven mutagenesis have been following. At the beginning, simple monogenic traits in the experimental condition have been elucidated. Then, more complex traits with variety of environmental interactions and gene-to-gene interactions （epistasis） have been challenged with mutant mice. In addition, chromosomal substitution strains and collaborative cross strains are also available to elucidate the complex traits in the mouse. Altogether, mouse models with mutagenesis and various laboratory strains will accelerate the studies of functional genomics in the mouse as well as in human.

关键词： mutagenesis functional genomics reverse genetics model animal quantitative trait epistasis

Rapid Recovery of Classical Swine Fever Virus Directly from Cloned cDNA

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Journal of Integrative Agriculture 2013年第5期12卷 877-883页

作者： HUANG Jun-hua LI Yong-feng HE Fan LI Dan SUN Yuan HAN Wen QIU Hua-ji Division of Swine Infectious Diseases State Key Laboratory of Veterinary BiotechnologyHarbin Veterinary Research InstituteChinese Academy of Agricultural Sciences

The reverse genetics for classical swine fever virus （CSFV） is currently based on the transfection of in vitro transcribed RNA from a viral genomic cDNA clone, which is inefficient and time-consuming. This study was aimed to develop an improved method for rapid recovery of CSFV directly from cloned cDNA. Full-length genomic cDNA from the CSFV Shimen strain, which was flanked by a T7 promoter, the hepatitis delta virus ribozyme and T7 terminator sequences, was cloned into the low- copy vector pOK12, producing pOKShimen-RzTФ. Direct transfection of pOKShimen-RzTqb into PK/T7 cells, a PK-15- derived cell line stably expressing bacteriophage T7 RNA polymerase, allowed CSFV to be rescued rapidly and efficiently, i.e., at least 12 h faster and 31.6-fold greater viral titer when compared with the in vitro transcription-based rescue system. Furthermore, the progeny virus rescued from PK/T7 cells was indistinguishable, both in vitro and in vivo, from its parent virus and the virus rescued from classical reverse genetics. The reverse genetics based on intracellular transcription is efficient, convenient and cost-effective. The PK/T7 cell line can be used to rescue CSFV directly from cloned cDNA and it can also be used as an intracellular transcription and expression system for studying the structure and function of viral genes.

关键词： classical swine fever virus reverse genetics T7 RNA polymerase stable cell line