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Construction of a subgenomic CV-B3 replicon expressing emerald green fluorescent protein to assess viral replication of a cardiotropic enterovirus strain in cultured human cells.

Coxsackieviruses B (CV-B) (Picornaviridae) are a common infectious cause of acute myocarditis in children and young adults, a disease, which is a precursor to 10-20% of chronic myocarditis and dilated cardiomyopathy (DCM) cases. The mechanisms involved in the disease progression from acute to chronic myocarditis phase and toward the DCM clinical stage are not fully understood but are influenced by both viral and host factors. Subgenomic replicons of CV-B can be used to assess viral replication mechanisms in human cardiac cells and evaluate the effects of potential antiviral drugs on viral repl…

A novel dengue virus inhibitor, BP13944, discovered by high-throughput screening with dengue virus replicon cells selects for resistance in the viral NS2B/NS3 protease.

Dengue virus (DENV) causes disease globally, resulting in an estimated 25 to 100 million new infections per year. No effective DENV vaccine is available, and the current treatment is only supportive. Thus, there is an urgent need to develop therapeutic agents to cure this epidemic disease. In the present study, we identified a potential small-molecule inhibitor, BP13944, via high-throughput screening (HTS) of 60,000 compounds using a stable cell line harboring an efficient luciferase replicon of DENV serotype 2 (DENV-2). BP13944 reduced the expression of the DENV replicon reporter in cells, sh…

Nipah virus-derived defective interfering particles generated using non-infectious viral replicon particles confer protective efficacy comparable to those produced with standard full-length infectious virus.

Defective interfering particles (DIPs) are naturally occurring virus-like particles containing truncated fragments of the parental viral gen … arrier to clinical translation. Here, we describe an improved, non-infectious, BSL-2-compatible NiV replicon system for DIP generation that eliminates the need for infectious virus. DIPs produced using this system inhibited virus in vitro and retained full protective efficacy in hamsters. By removing the requirement for high-containment virus, this approac …

A Minimal Replicon Enables Efficacious, Species-Specific Gene Deletion in Chlamydia and Extension of Gene Knockout Studies to the Animal Model of Infection Using Chlamydia muridarum.

The genus Chlamydia consists of diverse, obligate intracellular bacteria that infect various animals, including humans. Although chlamydial species share many aspects of the typical intracellular lifestyle, such as the biphasic developmental cycle and the preference for invasion of epithelial cells, each chlamydial strain also employs sophisticated species-specific strategies that contribute to an extraordinary diversity in organ and/or tissue tropism and disease manifestation. In order to discover and understand the mechanisms underlying how these pathogens infect particular hosts and cause s

Relevance of the N-terminal and major hydrophobic domains of non-structural protein 3A in the replicative process of a DNA-launched foot-and-mouth disease virus replicon.

A foot-and-mouth disease virus (FMDV) DNA-launched reporter replicon containing a luciferase gene was used to assess the impact of non-structural (NS) protein 3A on viral replication. Independent deletions within the N-terminal region (amino acid [aa] residues 6 to 24) and the central hydrophobic region (HR, aa 59 to 76) of FMDV NS protein 3A were engineered, and luciferase activity in lysates of control and mutated replicon-transfected cells was measured. Triple alanine replacements of the N-terminal triplet Arg 18- His 19 -Glu 20 and a single alanine substitution of the highly charged Glu 20…

Packaging the replicon RNA of the Far-Eastern subtype of tick-borne encephalitis virus into single-round infectious particles: development of a heterologous gene delivery system.

The sub-genomic replicon of tick-borne encephalitis (TBE) virus (Far-Eastern subtype) was packaged into infectious particles by providing the viral structural proteins in trans. Sequential transfection of TBE replicon RNA and a plasmid that expressed the structural proteins led to the secretion of infectious particles that contained TBE replicon RNA. The secreted particles had single-round infectivity, which was inhibited by TBE virus-neutralizing antibody. The physical structure of the particles was almost identical to that of infectious virions, and the packaged replicon RNA showed no recomb…

Диагностический потенциал и антигенные свойства рекомбинированных тикающих энцефалитных субвирусных частиц, выраженных в млекопитающих клетках из репликонов вируса Семликийского леса.

Протеины прекурсора (prME) всех трех подтипов вируса энцефалита (TBEV) были произведены на основе репликонов вируса Семлики (SFV), перебранных из рекомбинантных плазмидов. Клетки Vero E6, передающиеся этими плазмидами, продемонстрировали конкретную повторную активность в иммунофлюоресцентных и иммуноглотных анализах моноклонных антител против европейских и дальневосточных подтипов ТБЭВ, что свидетельствует о правильном сложении выраженных гликпротеинов. Гликопротеины ПРМЕ были скрыты в клеточной культуре, образуя подвирусные частицы от 20 до 30 нм в диаметре. На основе частиц prME Karelia-94 (

Совпадение в аннотации оригинала
The precursor membrane envelope (prME) proteins of all three tick-borne encephalitis virus (TBEV) subtypes were produced based on expression from Semliki Forest virus (SFV) replicons transcribed from recombinant plasmids. Vero E6 cells transfected by these plasmids showed specific reactivities in immunofluorescence and immunoblot assays by monoclonal antibod…

Репликоны рекомбинантной РНК, полученные в результате ослабленного венесуэльского энцефалитного вируса, защищают морских свиней и мышей от вируса геморрагической лихорадки Эболы.

Репликоны РНК, полученные в результате ослабления штамма венесуэльского энцефалита (VEE), альфавируса, были разработаны как потенциальные вакцины от геморрагической лихорадки Эболы. Гены Эболы нуклеопротеина (NP) или гликпротеина (GP) были введены в VEE RNA вниз по течению от VEE 26S промоутер вместо VEE структурно-белковых генов. Полученные рекомбинантные репликоны, выражающие гены NP или GP, были упакованы в репликонные частицы VEE (соответственно NP-VRP и GP-VRP) с использованием двухпартийной системы поддержки, которая обеспечивала структурные белки VEE в трансе и предотвратила регенерацию

Совпадение в аннотации оригинала
RNA replicons derived from an attenuated strain of Venezuelan equine encephalitis virus (VEE), an alphavirus, were configured as candidate vaccines for Ebola hemorrhagic fever. The Ebola nucleoprotein (NP) or glycoprotein (GP) genes were introduced into the VEE RNA downstream from the VEE 26S promoter in place of the VEE structural protein genes. The resulti…

[The interrelation between changes in the structural organization of replicon clusters, a retarded fork displacement rate and the high level of spontaneous SCEs in form II of xeroderma pigmentosum].

A cytogenetic observation, that the sister chromatid exchanges (SCE) occur 3 times more frequently in a special form of xeroderma pigmentosum--XPII than in the norm, prompted a study of DNA replication in this rare disease. Using DNA fiber autoradiography, the rate of fork movement and the frequency of initiation in the adjacent clusters of replicons were estimated. The rate of fork movement was significantly slower than that in classical XP and in normal cells. Here evidence was provided on another defect in DNA replication in XPII that involves a significantly decreased number of simultaneou…

Whole-genome analysis of a multidrug-resistant Klebsiella michiganensis environmental isolate from an orthopedic ward in Mwanza, Tanzania reveals IncF-family plasmid replicon signatures associated with resistance determinants.

BACKGROUND: Klebsiella michiganensis, a member of the Klebsiella oxytoca species complex, is increasingly recognized in healthcare-associate … I of 98.97% for A55848. The Tanzanian environmental isolate carried IncFIB(K) and IncFII(K) plasmid replicons, together with Col440I/Col440II. ResFinder-based screening identified a multidrug resistance gene repertoire including blaOXY family alleles (e.g., blaOXY-1-2), blaTEM-1B, oqxA/oqxB, aph(3')-Ia, aac(6')-Ib, and aadA2, consistent with an MDR genotyp …

Virulence genes and plasmid replicon profiles of selected β-lactamase-producing Acinetobacter baumannii from orthopaedic patients and the environment in a tertiary referral hospital in Tanzania, East Africa.

Acinetobacter baumannii has emerged as an important nosocomial pathogen due to its high resistance to multi-drugs and disinfectants plus its … underwent whole-genome sequencing (WGS) to detect sequence types (ST), β-lactamases genes, plasmid replicon types, and virulence genes. All four isolates harbored multiple β-lactamases genes including blaADC-25(3), blaOXA(4), blaCTX-M-15(2) and blaNDM-1(2). Furthermore, isolates harbored virulence genes encoding outer membrane protein (ompA), curli protei …

Preconceptual administration of an alphavirus replicon UL83 (pp65 homolog) vaccine induces humoral and cellular immunity and improves pregnancy outcome in the guinea pig model of congenital cytomegalovirus infection.

Development of a vaccine against congenital cytomegalovirus (CMV) infection is a major public health priority. We report the use of a propagation-defective, single-cycle, RNA replicon vector system, derived from an attenuated strain of the alphavirus Venezuelan equine encephalitis virus, to produce virus-like replicon particles (VRPs) expressing GP83, the guinea pig CMV (GPCMV) homolog of the human CMV pp65 phosphoprotein. Vaccination with VRP-GP83 induced antibodies and CD4(+) and CD8(+) T cell responses in GPCMV-seronegative female guinea pigs. Guinea pigs immunized with VRP-GP83 vaccine or …

Emerging threat of antimicrobial resistance determinants and plasmid replicon types acquisition by Escherichia coli of poultry and other food-producing animal origin in China: local findings with global implications.

Escherichia coli (E. coli) is a commensal and pathogenic bacterium responsible for harmless to severe infections in humans and food-producing animals inlcuding poultry. E. coli can significantly impact ecology and is a bioindicator of antimicrobial resistance (AMR) contamination. The global emergence of AMR is depleting the antimicrobial reserves for human use and highlights the need for antimicrobial stewardship. Generally, AMR emerges through the unjustified use of antibiotics in humans, food-producing animals, and agricultural settings. Animal species carry E. coli in their intestinal tract

Generation of VHSV replicon particles carrying transmembrane and C-terminal cytoplasmic region-deleted G gene (rVHSV-GΔTM) and comparison of vaccine efficacy with G gene-deleted VHSV (rVHSV-ΔG).

Vaccines based on viral replicon particles would be advantageous to induce immune responses compared to inactivated viruses in that they can infect host cells (only once) and can produce viral proteins in the infected cells like live viruses. Furthermore, as viral replicon particles are replication-defective, they are safer than live attenuated viruses. Previously, we had rescued viral hemorrhagic septicemia virus (VHSV) replicon particles lacking full ORF of G gene (rVHSV-ΔG). In the present study, to enhance the immunogenicity of VHSV replicon particles, we newly generated another form of VH…

Persistent inhibition of DNA synthesis in irradiated rat embryo fibroblasts expressing the oncogenes H-ras plus v-myc derives from inhibition of replicon initiation and is mitigated by staurosporine.

We have previously shown that rat embryo fibroblasts expressing the oncogenes H-ras plus v-myc experience a prolonged inhibition of DNA replication after exposure to ionizing radiation as compared to normal rat embryo fibroblasts, or rat embryo fibroblasts expressing H-ras or v-myc alone. Here we show that this enhanced inhibition of DNA replication in cells expressing H-ras plus v-myc is due to inhibition of the main controlling event of DNA replication, i.e., replicon initiation, that this inhibition is reversible, and that the expression of this phenotype is reverted by staurosporine, a pro…

Identification of an Arylnaphthalene Lignan Derivative as an Inhibitor against Dengue Virus Serotypes 1 to 4 (DENV-1 to -4) Using a Newly Developed DENV-3 Infectious Clone and Replicon.

Dengue virus (DENV) is the most widespread arbovirus, causing symptoms ranging from dengue fever to severe dengue, including hemorrhagic fever and shock syndrome. Four serotypes of DENV (DENV-1 to -4) can infect humans; however, no anti-DENV drug is available. To facilitate the study of antivirals and viral pathogenesis, here we developed an infectious clone and a subgenomic replicon of DENV-3 strains for anti-DENV drug discovery by screening a synthetic compound library. The viral cDNA was amplified from a serum sample from a DENV-3-infected individual during the 2019 epidemic; however, fragm…

Potent Anti-hepatitis C Virus (HCV) T Cell Immune Responses Induced in Mice Vaccinated with DNA-Launched RNA Replicons and Modified Vaccinia Virus Ankara-HCV.

Hepatitis C is a liver disease caused by the hepatitis C virus (HCV) affecting 71 million people worldwide with no licensed vaccines that prevent infection. Here, we have generated four novel alphavirus-based DNA-launched self-amplifying RNA replicon (DREP) vaccines expressing either structural core-E1-E2 or nonstructural p7-NS2-NS3 HCV proteins of genotype 1a placed under the control of an alphavirus promoter, with or without an alphaviral translational enhancer (grouped as DREP-HCV or DREP-e-HCV, respectively). DREP vectors are known to induce cross-priming and further stimulation of immune …

Dengue virus replicons: production of an interserotypic chimera and cell lines from different species, and establishment of a cell-based fluorescent assay to screen inhibitors, validated by the evaluation of ribavirin's activity.

The prevention and treatment of flavivirus infections are public health priorities. Dengue fever is the most prevalent mosquito-borne viral disease of humans, affecting more than 50 million people annually. Despite the urgent need to control dengue infections, neither specific antiviral therapies nor licensed vaccines exist and the molecular basis of dengue pathogenesis is not well understood. In this study we produced a novel dengue virus type 2 (DV2) subgenomic replicon that expresses a fusion protein comprised of Enhanced Green Fluorescent Protein (EGFP) and Puromycin N-Acetyltransferase (P…

Enhancement of the Replication of Hepatitis C Virus Replicons of Genotypes 1 to 4 by Manipulation of CpG and UpA Dinucleotide Frequencies and Use of Cell Lines Expressing SECL14L2 for Antiviral Resistance Testing.

Treatment for hepatitis C virus (HCV) has improved greatly through the use of direct-acting antivirals (DAAs). However, their effectiveness and potential for drug resistance development in non-genotype 1 variants of HCV remain relatively unexplored, as in vitro assays to assess drug susceptibility are poorly developed and unsuited for a transient-transfection format. In the current study, we have evaluated the effects of dinucleotide frequency changes in the replicon and the use of a SEC14L2-expressing cell line on the replication of HCVs of different genotypes and evaluated the resulting assa…

Endemic Indian clones of Klebsiella pneumoniae-harbouring New Delhi metallo-beta-lactamase-1 on a hybrid plasmid replicon type: A case of changing New Delhi metallo-beta-lactamase plasmid landscapes in India?

PURPOSE: blaNDM genes are MBL genes that confer resistance to carbapenems. Globally, they are associated with diverse clones and plasmids. In this study, we characterised three isolates of Klebsiella pneumoniae-harbouring blaNDM1 from patients undergoing chronic haemodialysis and renal transplantation. MATERIALS AND METHODS: 3 blaNDM1 -producing K. pneumoniae were isolated from end-stage renal disease patients undergoing haemodialysis and renal transplantation from a nephrology unit. All the three isolates were screened for clinically relevant resistant genes. Plasmid replicon content was ana…