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Marburg virus vaccines based upon alphavirus replicons protect guinea pigs and nonhuman primates.

Marburg virus (MBGV), for which no vaccines or treatments currently exist, causes an acute hemorrhagic fever with a high mortality rate in humans. We previously showed that immunization with either killed MBGV or a glycoprotein (GP) subunit prevented lethal infection in guinea pigs. In the studies reported here, an RNA replicon, based upon Venezuelan equine encephalitis (VEE) virus, was used as a vaccine vector; the VEE structural genes were replaced by genes for MBGV GP, nucleoprotein (NP), VP40, VP35, VP30, or VP24. Guinea pigs were vaccinated with recombinant VEE replicons (packaged into VE…

A strategy design based on antibiotic‑resistance and plasmid replicons genes of clinical Escherichia coli strains.

Since antimicrobial resistance, especially β-lactam resistance genes were common in clinical Escherichia coli strains, this study had designed and developed multiplex amplification platform for rapid and accurate detection of such resistance genes in 542 clinical E. coli isolates. The obtained specimens were subjected to bacteriological examination, antimicrobial susceptibility testing, and detection of β-lactamase genes and plasmid replicons. The major virulence genes were detected by 7 groups of multiplex PCR and eight groups of multiplex PCR were designed to detect 8 different plasmid repli…

The combined use of alphavirus replicons and pseudoinfectious particles for the discovery of antivirals derived from natural products.

Alphaviruses are a prominent class of reemergent pathogens due to their globally expanding ranges, potential for lethality, and possible use as bioweapons. The absence of effective treatments for alphaviruses highlights the need for innovative strategies to identify antiviral agents. Primary screens that use noninfectious self-replicating RNAs, termed replicons, have been used to identify potential antiviral compounds for alphaviruses. Only inhibitors of viral genome replication, however, will be identified using replicons, which excludes many other druggable steps in the viral life cycle. To …

Сооружение подгеномных репликонов желтой лихорадки с помощью метода гомологической рекомбинации дрожжей.

Репликон РНК, полученный из генома Флавивируса, является ценным инструментом для изучения вирусного репликации независимо от вирионной сборки и созревания, не говоря уже о большом потенциале для гетерологического генного выражения. В этом исследовании мы описали создание субгеномных репликонов вируса желтой лихорадки с помощью метода рекомбинации на дрожжевой основе. Плазмид с жёлтой лихорадкой 17D репликон штамма (pBSC-repYFV-17D), который ранее характеризовался, был обработан гетерологичным выражением генов зеленого флуоресцентного белка (repYFV-17D-GFP) и светлячка люциферазы (repYFV-17D-Lu

Совпадение в аннотации оригинала
RNA replicon derived from Flavivirus genome is a valuable tool for studying viral replication independent of virion assembly and maturation, besides being a great potential for heterologous gene expression. In this study we described the construction of subgenomic replicons of yellow fever virus by yeast-based homologous recombination technique. The plasmid …

Control of replication of FII plasmids: comparison of the basic replicons and of the copB systems of plasmids R100 and R1.

The copy numbers of the FII plasmids R1 and R100 were determined in four different ways and found to be identical. Deletion of one of the copy number control genes, copB, together with its promoter gives rise to plasmid copy mutants with an increased copy number. The increase was found to be 8- and 3.5-fold for plasmids R1 and R100, respectively. These deletion derivatives were found to be extremely sensitive to the presence of CopB activity from their own parent plasmid but not to that of the other plasmid. Hence, the CopB protein and its target are plasmid-specific and not FII-group-specific

Sindbis virus self-amplifying replicon is compatible with modified nucleotides mediating expression and vaccine responses in vivo.

The advent of effective formulations based on RNA encapsulated within lipid nanoparticles has led to the evaluation of this technology for i … ong-term expression of a protein is required. We generated self-amplifying RNA containing the viral replicon from Sindbis virus, which demonstrated robust protein expression in vitro and in vivo. In addition, it showed increased compatibility with a number of modified nucleotides when compared to an equivalent RNA containing the replicon from Venezuelan eq …

Potent therapeutic efficacy of an alphavirus replicon DNA vaccine expressing human papilloma virus E6 and E7 antigens.

Cervical cancer develops as a result of infection with high-risk human papillomavirus (HPV) through persistent expression of early proteins … h both SFV-based vaccines and DNA vaccines were combined with the development of a DNA-launched RNA replicon (DREP) vaccine targeting cervical cancer. Using intradermal delivery followed by electroporation, we demonstrated that DREP encoding for E6,7 (DREP-E6,7) induced effective, therapeutic antitumor immunity. While immunizations with a conventional DNA …

Replicon Particle Expressing the E2 Glycoprotein of Bovine Viral Diarrhea Virus Immunization and Evaluation of Antibody Response.

The objective of this study was to develop a new antigen delivery system using an alphavirus replicon particle (RP) to induce humoral antibody responses against bovine viral diarrhea virus (BVDV) recombinant antigen produced from envelope glycoprotein E2. An alphavirus RP expressing the E2 glycoprotein of BVDV was used for immunization of pigs. A fluorescent microsphere immunoassay (FMIA) has been applied to detect BVDV E2 antigen-specific antibody isotype in pig immunized with alphavirus RP. Full-length BVDV E2 (aa 1-375) was cleaved into several pieces, eight E2 DNA fragments, including full…

Venezuelan equine encephalitis virus replicon particle vaccine protects nonhuman primates from intramuscular and aerosol challenge with ebolavirus.

There are no vaccines or therapeutics currently approved for the prevention or treatment of ebolavirus infection. Previously, a replicon vaccine based on Venezuelan equine encephalitis virus (VEEV) demonstrated protective efficacy against Marburg virus in nonhuman primates. Here, we report the protective efficacy of Sudan virus (SUDV)- and Ebola virus (EBOV)-specific VEEV replicon particle (VRP) vaccines in nonhuman primates. VRP vaccines were developed to express the glycoprotein (GP) of either SUDV or EBOV. A single intramuscular vaccination of cynomolgus macaques with VRP expressing SUDV GP…

Development and characterization of a Rift Valley fever virus cell-cell fusion assay using alphavirus replicon vectors.

Rift Valley fever virus (RVFV), a member of the Phlebovirus genus in the Bunyaviridae family, is transmitted by mosquitoes and infects both … ly delivered to the cell surface. However, overexpression of RVFV glycoproteins using an alphavirus replicon vector resulted in the expression of the glycoproteins on the surface of multiple cell types. Brief treatment of RVFV glycoprotein expressing cells with mildly acidic media (pH 6.2 and below) resulted in rapid and efficient syncytia formation, which …

Bridging the antiviral gap: A BAC-based non-infectious SARS-CoV-2 replicon reveals potent antisense gapmers.

To facilitate studies of SARS-CoV-2 replication and antiviral drug discovery, we developed a non-infectious SARS-CoV-2 replicon, pBAC-SARS-CoV-2_S/eGFP_ΔE, based on a bacterial artificial chromosome vector. The viral genome was modified to ensure non-infectivity while allowing replication monitoring: the spike (S) protein and envelope (E) protein open reading frames (ORF) were deleted, and ORF S was replaced with an enhanced GFP (eGFP) reporter. Upon transfection, the pBAC-SARS-CoV-2_S/eGFP_ΔE was replication-competent in HEK293T cells and was inhibited by remdesivir, validating the replicon u…

An intranasally- and intramuscularly-deliverable nanostructured lipid carrier-replicon RNA vaccine drives protective systemic and mucosal immunity.

A nucleic acid vaccine platform that could be flexibly administered either intranasally or intramuscularly could be a valuable new tool for epidemic and pandemic response, combining rapid antigen adaptability with optimal induction of systemic and/or mucosal immune responses most appropriate for a particular pathogen. However, such RNA vaccines have not yet been developed. We have developed and optimized dual intranasal and intramuscular deliverability of a proof-of-concept replicon vaccine expressing an H5 influenza antigen that uses a nanostructured lipid carrier (NLC) delivery system. A rel…

A Newly Incompatibility F Replicon Allele (FIB81) in Extensively Drug-Resistant Escherichia coli Isolated from Diseased Broilers.

Multiple drug resistance (MDR) has gained pronounced attention among Enterobacterales. The transfer of multiple antimicrobial resistance gen … Salmonella spp. and E. coli isolated from broilers. The antimicrobial resistance profile, PCR-based replicon typing of bacterial isolates pre- and post-plasmid curing, and IncF replicon allele sequence typing were investigated. Five isolates of E. coli (5/31; 16.13%) and Salmonella spp. (5/36; 13.89%) were pan-susceptible to the examined antimicrobial agen …

A Unique Robust Dual-Promoter-Driven and Dual-Reporter-Expressing SARS-CoV-2 Replicon: Construction and Characterization.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2, SARS2) remains a great global health threat and demands identification of more effective and SARS2-targeted antiviral drugs, even with successful development of anti-SARS2 vaccines. Viral replicons have proven to be a rapid, safe, and readily scalable platform for high-throughput screening, identification, and evaluation of antiviral drugs against positive-stranded RNA viruses. In the study, we report a unique robust HIV long terminal repeat (LTR)/T7 dual-promoter-driven and dual-reporter firefly luciferase (fLuc) and green fluor…

Heterologous protection against Crimean-Congo hemorrhagic fever in mice after a single dose of replicon particle vaccine.

No vaccines are currently licensed to prevent Crimean-Congo hemorrhagic fever virus (CCHFV) infection, which can cause mild self-limiting clinical signs or severe, often fatal hemorrhagic fever disease. Here we continued investigations into the utility of a single-dose virus replicon particle (VRP) vaccine regimen by assessing protection against Turkey or Oman strains of CCHFV. We found that all mice were completely protected from disease, supporting broad applicability of this platform for CCHFV prevention.

Development of a replicon cell line-based high throughput antiviral assay for screening inhibitors of Zika virus.

Zika virus (ZIKV) is an important emerging human pathogen associated with microcephaly, Guillain-Barré syndrome and meningoencephalitis. Developing rapid and reliable HTS assay is important for ZIKV drug discovery. Here, we constructed a dicistronic ZIKV replicon (ZIKV-Pac-Rluc-Rep) that contained the Renilla luciferase (Rluc) reporter gene separated from the puromycin N-acetyl-transferase (Pac) selectable marker by a short peptide cleavage site. A clonal replicon cell line stably expressing high level of ZIKV replicon was established by selection with puromycin. By optimizing cell number, com…

Mosquito cell-derived West Nile virus replicon particles mimic arbovirus inoculum and have reduced spread in mice.

Half of the human population is at risk of infection by an arthropod-borne virus. Many of these arboviruses, such as West Nile, dengue, and … d by arthropods, arboviruses should be derived from arthropod cells. Previous studies have packaged replicon genome in mammalian cells to produce replicon particles, which undergo only one round of infection, but no studies exist packaging replicon particles in mosquito cells. Here we optimized the packaging of West Nile virus replicon genome in mosquito c …

Replicon typing of plasmids carrying bla CTX-M-1 in Enterobacteriaceae of animal, environmental and human origin.

OBJECTIVES: The aim of this work was to determine the plasmid replicon profiles of a collection of bla CTX-M-1-positive enterobacterial strains. The isolates originated from chicken in the production pyramid, healthy food-producing animals at slaughter (chicken, calves, and pigs), chicken retail meat, environmental isolates originating from water bodies, and isolates from humans. A selection of IncI and IncN plasmids were characterized by multilocus sequence typing in order to determine their epidemiological relatedness. METHODS: Transconjugants of 74 bla CTX-M-1-positive isolates were analyz…

Novel vancomycin-resistance transposon, plasmid replicon types, and virulence factors of vancomycin-resistant Enterococci in Zhejiang, China.

Forty-seven vancomycin-resistant Enterococcus (VRE) strains were isolated from clinical samples in 13 Zhejiang hospitals and fecal samples f … intergenic region of Tn1546 leading to coresistance to vancomycin and fosfomycin. The four plasmid replicon types (pRUM, pRE25, pEF418, and pB82) were more common in VRE isolates, suggesting their association with vancomycin resistance and nosocomial transmission. The prevalence rate of vancomycin-resistant Staphylococcus aureus-related Inc18-like plasmid …

Single-dose immunization with virus replicon particles confers rapid robust protection against Rift Valley fever virus challenge.

Rift Valley fever virus (RVFV) causes outbreaks of severe disease in people and livestock throughout Africa and the Arabian Peninsula. The potential for RVFV introduction outside the area of endemicity highlights the need for fast-acting, safe, and efficacious vaccines. Here, we demonstrate a robust system for the reverse genetics generation of a RVF virus replicon particle (VRP(RVF)) vaccine candidate. Using a mouse model, we show that VRP(RVF) immunization provides the optimal balance of safety and single-dose robust efficacy. VRP(RVF) can actively synthesize viral RNA and proteins but lacks…