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Rubella virus-like replicon particles: analysis of encapsidation determinants and non-structural roles of capsid protein in early post-entry replication.

Rubella virus (RUBV) contains a plus-strand RNA genome with two ORFs, one encoding the non-structural replicase proteins (NS-ORF) and the second encoding the virion structural proteins (SP-ORF). This study describes development and use of a trans-encapsidation system for the assembly of infectious RUBV-like replicon particles (VRPs) containing RUBV replicons (self replicating genomes with the SP-ORF replaced with a reporter gene). First, this system was used to map signals within the RUBV genome that mediate packaging of viral RNA. Mutations within a proposed packaging signal did not significa…

Identification of the corticotropin-releasing factor receptor 1 antagonists as inhibitors of Chikungunya virus replication using a Gaussia luciferase-expressing subgenomic replicon.

The Chikungunya virus (CHIKV), an enveloped RNA virus that has been identified in over 40 countries and is considered a growing threat to public health worldwide. However, there is no preventive vaccine or specific therapeutic drug for CHIKV infection. To identify a new inhibitor against CHIKV infection, this study constructed a subgenomic RNA replicon expressing the secretory Gaussia luciferase (Gluc) based on the CHIKV SL11131 strain. Transfection of in vitro-transcribed replicon RNA to BHK-21 cells revealed that Gluc activity in culture supernatants was correlated with the intracellular rep…

Phase I safety and immunogenicity evaluations of an alphavirus replicon HIV-1 subtype C gag vaccine in healthy HIV-1-uninfected adults.

On the basis of positive preclinical data, we evaluated the safety and immunogenicity of an alphavirus replicon HIV-1 subtype C gag vaccine (AVX101), expressing a nonmyristoylated form of Gag, in two double-blind, randomized, placebo-controlled clinical trials in healthy HIV-1-uninfected adults. Escalating doses of AVX101 or placebo were administered subcutaneously to participants in the United States and Southern Africa. Because of vaccine stability issues, the first trial was halted prior to completion of all dose levels and a second trial was implemented. The second trial was also stopped p…

Hepatitis C virus nucleotide inhibitors PSI-352938 and PSI-353661 exhibit a novel mechanism of resistance requiring multiple mutations within replicon RNA.

PSI-352938, a cyclic phosphate nucleotide, and PSI-353661, a phosphoramidate nucleotide, are prodrugs of β-D-2'-deoxy-2'-α-fluoro-2'-β-C-methylguanosine-5'-monophosphate. Both compounds are metabolized to the same active 5'-triphosphate, PSI-352666, which serves as an alternative substrate inhibitor of the NS5B RNA-dependent RNA polymerase during HCV replication. PSI-352938 and PSI-353661 retained full activity against replicons containing the S282T substitution, which confers resistance to certain 2'-substituted nucleoside/nucleotide analogs. PSI-352666 was also similarly active against both …

DNA-Launched Alphavirus Replicons Encoding a Fusion of Mycobacterial Antigens Acr and Ag85B Are Immunogenic and Protective in a Murine Model of TB Infection.

There is an urgent need for effective prophylactic measures against Mycobacterium tuberculosis (Mtb) infection, particularly given the highl … degrees of success. In the present study, we evaluated DNA-launched Venezuelan equine encephalitis replicons (Vrep) encoding a novel fusion of the mycobacterial antigens α-crystallin (Acr) and antigen 85B (Ag85B), termed Vrep-Acr/Ag85B, for their immunogenicity and protective efficacy in a murine model of pulmonary TB. Vrep-Acr/Ag85B generated antigen-spe …

Analysis of the effects of alterations in the tick-borne encephalitis virus 3'-noncoding region on translation and RNA replication using reporter replicons.

The 3'-noncoding region (3'-NCR) of the flavivirus genome includes a variable region that tolerates the insertion of heterologous genetic information. Natural isolates of tick-borne encephalitis virus (TBEV) have particularly long variable regions, which, for some strains, include an internal poly(A) tract. We constructed luciferase reporter replicons of TBEV to analyze the impact of various manipulations of the 3'-NCR on viral RNA translation and replication. The choice of the reporter gene, its position and processing within the viral polyprotein, and the choice of standards were found to be…

Construction and characterization of Genotype-3 hepatitis C virus replicon revealed critical genotype-3-specific polymorphism for drug resistance and viral fitness.

Hepatitis C virus (HCV), a major causative agent of chronic hepatitis, is a positive-stranded RNA virus and has a high degree of genetic div … rlying molecular mechanisms remain to be explored. Here we developed a novel genotype-3a subgenomic replicon PR87A7 by screening a HCV cDNA pool amplified from a patient serum RNA. PR87A7 replicon displayed strong resistance to anti-NS3 DAAs, mainly owing to a genotype-3-specific polymorphism 168Q in NS3. Introduction of NS3 168Q into a genotype-2a JFH1 st …

PTC725, an NS4B-Targeting Compound, Inhibits a Hepatitis C Virus Genotype 3 Replicon, as Predicted by Genome Sequence Analysis and Determined Experimentally.

PTC725 is a small molecule NS4B-targeting inhibitor of hepatitis C virus (HCV) genotype (gt) 1 RNA replication that lacks activity against HCV gt2. We analyzed the Los Alamos HCV sequence database to predict susceptible/resistant HCV gt's according to the prevalence of known resistance-conferring amino acids in the NS4B protein. Our analysis predicted that HCV gt3 would be highly susceptible to the activity of PTC725. Indeed, PTC725 was shown to be active against a gt3 subgenomic replicon with a 50% effective concentration of ∼5 nM. De novo resistance selection identified mutations encoding am…

A phase I dose escalation trial of vaccine replicon particles (VRP) expressing prostate-specific membrane antigen (PSMA) in subjects with prostate cancer.

PSMA-VRP is a propagation defective, viral replicon vector system encoding PSMA under phase I evaluation for patients with castration resistant metastatic prostate cancer (CRPC). The product is derived from an attenuated strain of the alphavirus, Venezuelan Equine Encephalitis (VEE) virus, and incorporates multiple redundant safety features. In this first in human trial, two cohorts of 3 patients with CRPC metastatic to bone were treated with up to five doses of either 0.9×10(7)IU or 0.36×10(8)IU of PSMA-VRP at weeks 1, 4, 7, 10 and 18, followed by an expansion cohort of 6 patients treated wit…

Construction of Fosmid-based SARS-CoV-2 replicons for antiviral drug screening and replication analyses in biosafety level 2 facilities.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has necessitated the global development of countermeasures since its outbreak. However, current therapeutics and vaccines to stop the pandemic are insufficient and this is mainly because of the emergence of resistant variants, which requires the urgent development of new countermeasures, such as antiviral drugs. Replicons, self-replicating RNAs that do not produce virions, are a promising system for this purpose because they safely recreate viral replication, enabling antiv…

Efficacy of Western, Eastern, and Venezuelan Equine Encephalitis (WEVEE) Virus-Replicon Particle (VRP) Vaccine against WEEV in a Non-Human Primate Animal Model.

The purpose of this study was to evaluate the effects of the route of administration on the immunogenicity and efficacy of a combined western, eastern, and Venezuelan equine encephalitis (WEVEE) virus-like replicon particle (VRP) vaccine in cynomolgus macaques. The vaccine consisted of equal amounts of WEEV, EEEV, and VEEV VRPs. Thirty-three animals were randomly assigned to five treatment or control groups. Animals were vaccinated with two doses of WEVEE VRPs or the control 28 days apart. Blood was collected 28 days following primary vaccination and 21 days following boost vaccination for ana…

Virus replicon particles expressing porcine reproductive and respiratory syndrome virus proteins elicit immune priming but do not confer protection from viremia in pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of one of the most devastating and economically significant viral disease of pigs worldwide. The vaccines currently available on the market elicit only limited protection. Recombinant vesicular stomatitis virus (VSV) replicon particles (VRP) have been used successfully to induce protection against influenza A virus (IAV) in chickens and bluetongue virus in sheep. In this study, VSV VRP expressing the PRRSV envelope proteins GP5, M, GP4, GP3, GP2 and the nucleocapsid protein N, individually or in combination, wer…

Construction of an infectious molecular clone of Japanese encephalitis virus genotype V and its derivative subgenomic replicon capable of expressing a foreign gene.

Japanese encephalitis virus (JEV) genotype V was originally isolated in Malaysia in 1952 and has long been restricted to the area. In 2009, … ranscribed in vitro from the modified plasmid was shown to replicate itself in mammalian cells as a replicon. The replicon carrying the firefly luciferase gene in place of the deleted structural protein genes was also shown to efficiently replicate itself and express luciferase in mammalian cells. Compared with the replicon derived from JEV genotype III (N …

Гетерологический венесуэльский энцефалитный репликонный репликонный режим вакцины денге обеспечивает полную защиту от вирусного вызова в циномольгусе.

Потенциальная вакцина (D1ME-VRP), содержащая вирус денге типа 1 премембрана и белки конвертов в венесуэльской системе репликонных частиц (VRP), была построена и испытана в сочетании с плазмидной вакциной ДНК (D1ME-DNA), содержащей идентичные последовательности вирусов денге. Циномольгузные макаки были привиты тремя дозами ДНК (ДДД), тремя дозами ВРП (группа VVV) или гетерологическим редукционным режимом ДНК (DDV) с использованием двух доз вакцины ДНК и третьей дозы вакцины ВРП. Через четыре недели после окончательной иммунизации группа DDV произвела самые высокие вирусные вирусы денге типа 1,

Совпадение в аннотации оригинала
A candidate vaccine (D1ME-VRP) expressing dengue virus type 1 premembrane and envelope proteins in a Venezuelan equine encephalitis (VEE) virus replicon particle (VRP) system was constructed and tested in conjunction with a plasmid DNA vaccine (D1ME-DNA) expressing identical dengue virus sequences. Cynomolgus macaques were vaccinated with three doses of DNA …

Evaluation of Venezuelan Equine Encephalitis (VEE) replicon-based Outer surface protein A (OspA) vaccines in a tick challenge mouse model of Lyme disease.

Venezuelan Equine Encephalitis (VEE) virus replicon particles (VRPs) encoding Borrelia burgdorferi Outer surface protein A (OspA) were evaluated for their ability to induce an immune response and provide protection from tick-borne spirochetes. VRPs expressing ospA that accumulated intracellularly (VRP OspA) or that was secreted from host cells (VRP tPA-OspA) were tested. Both VRP OspA and VRP tPA-OspA expressed ospA in immunized mice. Mice vaccinated with VRPs expressing secreted OspA produced significant amounts of anti-OspA antibodies, whereas VRPs expressing intracellular OspA were less imm…

Antimicrobial susceptibility, plasmid replicon typing, phylogenetic grouping, and virulence potential of avian pathogenic and faecal Escherichia coli isolated from meat chickens in Australia.

Globally, avian colibacillosis is a leading cause of morbidity and mortality in poultry, associated with economic losses and welfare problems. Here, clinical avian pathogenic E. coli isolates (CEC; n = 50) and faecal E. coli isolates from healthy (FEC; n = 187) Australian meat chickens collected between 2006 and 2014 were subjected to antimicrobial susceptibility testing, phylogenetic grouping, plasmid replicon (PR) typing, multilocus sequence typing, and virulence gene (VG) profiling. Extended-spectrum cephalosporin (ESC)- and fluoroquinolone (FQ)-resistant E. coli isolates underwent further …

Development of a novel Dengue-1 virus replicon system expressing secretory Gaussia luciferase for analysis of viral replication and discovery of antiviral drugs.

Replicon systems have been used for high-throughput screening of anti-dengue virus (anti-DENV) inhibitors and for understanding mechanisms of viral replication. In the present study, we constructed novel DENV-1 replicons encoding Gaussia luciferase that was secreted into the culture medium. Two types of constructs were generated: RNA-based and DNA-based. Each type was translated in an internal ribosome entry site (IRES)-dependent or IRES-independent manner. Among these constructs, the DNA-based replicon employing IRES-dependent translation (DGL2) produced the highest titer. Luciferase levels i…

A sindbis virus replicon-based DNA vaccine encoding the rabies virus glycoprotein elicits immune responses and complete protection in mice from lethal challenge.

A sindbis virus replicon-based DNA vaccine encoding rabies virus glycoprotein (G) was developed by subcloning rabies G gene into a sindbis virus replicon-based vaccine vector (pAlpha). The self-amplification of RNA transcripts and translation efficiency of rabies G was analyzed in pAlpha-Rab-G-transfected mammalian cells using RT-PCR, SDS-PAGE and Western blot analysis. The transfected cells also showed induction of apoptosis which is an important event in the enhancement of immune responses. Further, immune responses induced with replicon-based rabies DNA vaccine (pAlpha-Rab-G) was compared w…

Inhibition of mammalian deoxyribonucleic acid synthesis by neocarzinostatin: selective effect on replicon initiation in CHO cells and resistant synthesis in ataxia telangiectasia fibroblasts.

Treatment of CHO cells with low doses of the protein antibiotic neocarzinostatin severely inhibited DNA replicon initiation but had no effect on chain elongation. The selectivity of the effect on initiation, which was greater than that seen with other chemical agents and comparable to that seen with X-rays, explains the biphasic dose response seen for DNA synthesis inhibition by this drug. Parallel experiments employing the nucleoid sedimentation technique indicated that half-maximal relaxation of domains of DNA supercoiling and half-maximal inhibition of replicon initiation required the same …

WGS-Based Phenotyping and Molecular Characterization of the Resistome, Virulome and Plasmid Replicons in Klebsiella pneumoniae Isolates from Powdered Milk Produced in Germany.

The emergence of Klebsiella pneumoniae (K. pneumoniae) in German healthcare is worrying. It is not well-investigated in the veterinary world and food chains. In the current study, antibiotic susceptibility profiles of 24 K. pneumoniae strains isolated from powdered milk samples produced in Germany were investigated by a microdilution test. Next-generation sequencing (NGS) was applied to identify genomic determinants for antimicrobial resistance (AMR), virulence-associated genes and plasmids replicons. All isolates were susceptible to the majority (14/18) of tested antibiotics. Resistance to co…