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PlasmidFinder and in silico plasmid multiLocus sequence typing (pMLST) are two easy-to-use web tools for detection and characterization of plasmid sequences in whole-genome sequencing (WGS) data from Enterobacteriaceae. These tools have been adopted worldwide and facilitate plasmid detection and typing based on draft genomes of multi-drug-resistant Enterobacteriaceae. The PlasmidFinder database currently includes 133 unique plasmid replicon sequences. It was built starting with 126 sequences devised on fully sequenced plasmids available at the NCBI nucleotide database in 2014 and has been continuously updated to include novel replicons detected in more recently sequenced plasmids associated with the family Enterobacteriaceae. PlasmidFinder is usable for replicon sequence analysis of raw as well as assembled sequencing data. For pMLST analysis, a weekly updated database was generated from www.pubmlst.org and integrated into a web tool called in silico pMLST.

Alphaviruses have been used as vectors for the expression of heterologous genetic information for a few decades. Their genomes contain two open reading frames. One of these encodes viral nonstructural proteins (nsPs), which mediate the replication of viral genome (G RNA) and synthesis of subgenomic (SG) RNA. Viral structural proteins encoded by SG RNA are dispensable for G RNA replication and transcription of the SG RNA. Their genes can be replaced by heterologous genetic materials. Such defective viral genomes, replicons, can efficiently express the encoded heterologous genes of interest. Within recent years, alphavirus replicons, also termed self-amplifying RNAs (saRNAs), attracted additional attention as a tool to increase the expression level of mRNA vaccines. The results of this study demonstrate that replicons derived from both New World and Old World alphaviruses can efficiently express heterologous genetic information. However, the levels of expression are cell-specific and are strongly determined by the origin of the replicons. Moreover, replication of alphavirus replicons mimics many aspects of alphavirus infections in terms of virus-host interactions and induction of the innate immune response. They also express viral nsPs that may induce vector immunity. These characteristics need to be carefully examined before the wide application of saRNAs. On the other hand, the data show that alphavirus replicons are very flexible expression systems. The efficiencies of protein expression and interactions with host cells can be manipulated by introducing mutations into nsPs and other modifications in replicon genomes. [ABSTRACT FROM AUTHOR]

The next step in mRNA vaccine design is the application of viral-based self-amplifying mRNAs (replicons) that provide long-lasting humoral and cellular immune responses upon single, low-dose immunization. Replicons encode their own replication machinery to boost their copy numbers directly after administration in target cells, which dramatically lowers the required initial mRNA dose and may consequently reduce adverse effects in individuals. Recent advances in mRNA formulation using lipid or solid nanoparticles create opportunities for novel applications for replicons such as mucosal delivery. Replicon vaccines hold potential as a platform technology when safety aspects are properly addressed. A diverse spectrum of replicons have been developed for innovative applications such as multivalent and therapeutic cancer vaccines. mRNA vaccines have won the race for early COVID-19 vaccine approval, yet improvements are necessary to retain this leading role in combating infectious diseases. A next generation of self-amplifying mRNAs, also known as replicons, form an ideal vaccine platform. Replicons induce potent humoral and cellular responses with few adverse effects upon a minimal, single-dose immunization. Delivery of replicons is achieved with virus-like replicon particles (VRPs), or in nonviral vehicles such as liposomes or lipid nanoparticles. Here, we discuss innovative advances, including multivalent, mucosal, and therapeutic replicon vaccines, and highlight novelties in replicon design. As soon as essential safety evaluations have been resolved, this promising vaccine concept can transform into a widely applied clinical platform technology taking center stage in pandemic preparedness. [ABSTRACT FROM AUTHOR]

Background: pTE15 is a ~ 15-kb narrow-host-range indigenous plasmid from Lactobacillus reuteri N16 that does not replicate in selected Bacillus spp., Staphylococcus spp., and other Lactobacillus spp. Methods: Combined deletion analysis the minireplicon essential of pTE15 with replicon-probe vector pUE80 (−) to confirmed sufficient for replication and from the ssDNA intermediate detection, plasmid amplification tested by chloramphenicol treatment, and replication origin sequence analysis to delineated the novel theta-type replication of pTE15. Results: Single-stranded intermediate of pTE15 DNA was not detected in L. reuteri, indicating that this plasmid does not replicate via a rolling circle mechanism. The replicon of pTE15 did not display the structural organization typical of rolling-circle plasmids, nor were they similar to known rolling-circle plasmids. We further provided evidence that this plasmid applied a new mode of theta-type replication mechanism: (1) the size of this plasmid was > 10-kb; (2) the minireplicon consisted of AT-rich (directed repeat, iteron) and DnaA sequences; (3) the minireplicon did not contain double-strand origin (DSO) and essential rep genes, and it also showed no single-strand origin (SSO) structure; (4) the intermediate single-stranded DNA products were not observed for pTE15 replication; (5) the minireplicon did not contain a typical essential replication protein, Rep, (6) its copy number was decreased by chloramphenicol treatment, and (7) genes in pTE15 replication region encoded truncated RepA (TRepA), RepB and RepC, which were replication-associated proteins, but they were not essential for pTE15 replication. Conclusions: Collectively, our results strongly suggested that the indigenous plasmid pTE15 of L. reuteri N16 belongs to a new class of theta replicons. [ABSTRACT FROM AUTHOR]

Self-amplifying (sa) RNA molecules—“replicons”—derived from the genomes of positive-sense RNA viruses are receiving increasing attention as gene and vaccine delivery vehicles. This is because mRNA forms of genes of interest can be incorporated into them and strongly amplified, thereby enhancing target protein expression. In this report, we demonstrate a nonmonotonic dependence of protein expression on the mass of transfected replicon, in contrast to the usual, monotonic case of non-saRNA transfections. We lipotransfected a variety of cell lines with increasing masses of enhanced yellow fluorescent protein (eYFP) as a reporter gene in sa form and found that there is a “sweet spot” at which protein expression and cell viability are optimum. To control the varying mass of transfected replicon RNA for a given mass of Lipofectamine, the replicons were mixed with a “carrier” RNA that is neither replicated nor translated; the total mass of transfected RNA was kept constant while increasing the fraction of the replicon from zero to one. Fluorescence microscopy studies showed that the optimum protein expression and cell viability are achieved for replicon fractions as small as 1/10 of the total transfected RNA, and these results were quantified by a systematic series of flow cytometry measurements. IMPORTANCE Positive-sense RNA viruses often have a cytotoxic effect on their host cell because of the strength of their RNA replicase proteins, even though only one copy of their genome begins the viral life cycle in each cell. Noninfectious forms of them—replicons—which include just their RNA replication-related genes, are also strongly self-amplifying and cytotoxic. Accordingly, when replicons fused with nonviral genes of interest are transfected into cells to amplify expression of proteins of interest, one needs to keep the replicon “dose” sufficiently low. We demonstrate how to control the number of RNA replicons getting into transfected cells and that there is a sweet spot for the replicon dose that optimizes protein expression and cell viability. Examples are given for the case of Nodamura viral replicons with fluorescent protein reporter genes in a variety of mammalian cell lines, quantified by flow cytometry and live/dead cell assays. [ABSTRACT FROM AUTHOR]

To evaluate avian hepatitis E virus (aHEV) as an RNA vaccine platform, ORF2 of aHEV was replaced by heterologous genes, such as eGFP and HA-tag, in aHEV infectious cDNA clones. eGFP and HA-tag replicons were expressed in LMH cells. To confirm expression of the heterologous protein, ORF2 was replaced with the antigenic S1 gene of IBV. The IBVS1 replicon was expressed in LMH cells. To our knowledge, this is the first investigation showing the potential as a RNA vaccine platform using an aHEV. In the future, it may be used in the development of RNA vaccines against various pathogens. [ABSTRACT FROM AUTHOR]

The development of direct-acting antivirals (DAAs) has revolutionized the state-of-the art treatment of HCV infections, with sustained virologic response rates above 90%. However, viral variants harboring substitutions referred to as resistance-associated substitutions (RASs) may be present in baseline levels and confer resistance to DAAs, thereby posing a major challenge for HCV treatment. HCV replicons have been the primary tools for discovering and evaluating the inhibitory activity of DAAs against viral replication. Interest in replicon systems has further grown as they have become indispensable for discovering genotype-specific and cross-genotype RASs. Here, we review functional replicon systems for HCV, how these replicon systems have contributed to the development of DAAs, and the characteristics and distribution of RASs for DAAs. [ABSTRACT FROM AUTHOR]

Unlike human isolates, environmental Escherichia coli isolates have not been thoroughly investigated for the diversity and transferability of antibiotic-resistant plasmids. In this study, antibiotic-resistant strains from marine sediment (n = 50) and clams (n = 53) were analyzed (i) for their plasmid content using a PCR-based plasmid replicon typing (PBRT) kit and (ii) for the transferability of plasmid-associated antibiotic resistance (AR) traits by mating experiments. Fifteen of the thirty replicons targeted by the PBRT kit were detected in the isolates; 8/15 were identified in both sediment and clam isolates, although at different frequencies. The most frequent replicons in sediment (74%) and in clam strains (66%) alike, were FIA, FIB, or FII, which are associated with the IncF group, followed by the I1α replicon, which was more frequent in clam (24.5%) than in sediment (10%) strains. More than 50% of the strains contained multiple replicons; although 15 were untypable, S1-PFGE analysis demonstrated that 14/15 carried no plasmids. All cryptic strains were successfully typed and were positive for IncF or IncI replicons. Antibiotic-resistant strains accounted for 63% of all isolates and were significantly (p < 0.05) more frequent in phylogroup A. Most (35%) multidrug-resistant (MDR) strains belonged to phylogroup A, too. Although 25/26 MDR strains were positive for IncF plasmids (the exception being a clam strain), the FII-FIB rep combination was predominant (63%) among the sediment isolates, whereas most clam isolates (40%) carried the FII replicon alone. In mating experiments, selected MDR strains carrying FIB, FII, and I1α replicons, used as the donors, transferred multiple ARs together with the IncF or IncI plasmids at high frequency. Since IncI plasmids are common in E. coli and Salmonella enterica isolates from poultry, our findings suggest an animal origin to the E. coli clam strains carrying IncI plasmids. They also suggest a role for IncI plasmids in the spread of ARs among environmental Enterobacteriaceae and, through the food chain, to human isolates. In conclusion, the PBRT kit proved to be a useful tool to identify plasmids carrying antibiotic-resistant genes and to shed light on the factors underpinning their diffusion. [ABSTRACT FROM AUTHOR]

Animal hepaciviruses represent promising surrogate models for hepatitis C virus (HCV), for which there are no efficient immunocompetent animal models. Experimental infection of laboratory rats with rodent hepacivirus isolated from feral Rattus norvegicus (RHV-rn1) mirrors key aspects of HCV infection in humans, including chronicity, hepatitis, and steatosis. Moreover, RHV has been adapted to infect immunocompetent laboratory mice. RHV in vitro systems have not been developed but would enable detailed studies of the virus life cycle crucial for designing animal experiments to model HCV infection. Here, we established efficient RHV-rn1 selectable subgenomic replicons with and without reporter genes. Rat and mouse liverderived cells did not readily support the complete RHV life cycle, but repliconcontaining cell clones could be selected with and without acquired mutations. Replication was significantly enhanced by mutations in NS4B and NS5A and in cell clones cured of replicon RNA. These mutations increased RHV replication of both mono- and bicistronic constructs, and CpG/UpA-dinucleotide optimization of reporter genes allowed replication. Using the replicon system, we show that the RHVrn1 NS3-4A protease cleaves a human mitochondrial antiviral signaling protein reporter, providing a sensitive readout for virus replication. RHV-rn1 replication was inhibited by the HCV polymerase inhibitor sofosbuvir and high concentrations of HCV NS5A antivirals but not by NS3 protease inhibitors. The microRNA-122 antagonist miravirsen inhibited RHV-rn1 replication, demonstrating the importance of this HCV host factor for RHV. These novel RHV in vitro systems will be useful for studies of tropism, molecular virology, and characterization of virus-host interactions, thereby providing important complements to in vivo systems. [ABSTRACT FROM AUTHOR]

Chronic hepatitis C virus (HCV) infections affect 71 million people worldwide, often resulting in severe liver damage. Since 2014 highly efficient therapies based on directly acting antivirals (DAAs) are available, offering cure rates of almost 100%, if the infection is diagnosed in time. It took more than a decade to discover HCV in 1989 and another decade to establish a cell culture model. This review provides a personal view on the importance of HCV cell culture models, particularly the replicon system, in the process of therapy development, from drug screening to understanding of mode of action and resistance, with a special emphasis on the contributions of Ralf Bartenschlager's group. It summarizes the tremendous efforts of scientists in academia and industry required to achieve efficient DAAs, focusing on the main targets, protease, polymerase and NS5A. It furthermore underpins the importance of strong basic research laying the ground for translational medicine. [ABSTRACT FROM AUTHOR]

Objectives: To analyse OXA-48 (OXA-48/181)-type carbapenemase-producing Enterobacteriaceae reported in Poland from 2013 until January 2017.Methods: Bacterial isolates were typed by PFGE and MLST. Genes coding for OXA-48/181 types and other β-lactamases were amplified and sequenced. Mobile elements with blaOXA-48/181-like genes were PCR mapped. blaOXA-48/181-carrying plasmids were characterized by nuclease S1-hybridization profiling, transfer assays and PCR-based replicon typing, while the chromosomal location of the genes was confirmed by the I-CeuI analysis.Results: Fifty-four isolates from 52 patients in 20 hospitals (14 cities) were included, in 14 cases having probable foreign origins indicated. The organisms were genetically diverse and represented numerous pandemic clones, including Klebsiella pneumoniae ST395 (n = 23), ST11, ST15 and ST101, Escherichia coli ST38, ST410 and ST648, and Enterobacter cloacae complex ST78. These produced OXA-48 (n = 49), OXA-181 (n = 4) or OXA-232 (n = 1). One of five K. pneumoniae ST395 pulsotypes caused a multicentre outbreak with 18 cases, which significantly contributed to the total number of patients. Depending on the variant, the blaOXA-48/181-like genes were parts of the Tn1999-, Tn2013- or Tn2016-like transposons, with blaOXA-48 found in an ISEcp1-associated module (Tn2016-like) for the first time. Three genotypes, including E. coli ST38, had chromosomal blaOXA-48 genes, while others carried transmissible IncL (∼60 kb; blaOXA-48; n = 30), IncM (∼80-95 kb; blaOXA-48; n = 4), IncX3 (∼50 kb; blaOXA-181; n = 4) or non-typeable (∼90-160 kb; blaOXA-48 or blaOXA-232) plasmids.Conclusions: Even though OXA-48/181 producers seem to occur infrequently in Poland, their epidemiology has been marked by various phenomena, namely multiple imports, several limited transmissions plus one larger clonal outbreak, and possible plasmid transfers. [ABSTRACT FROM AUTHOR]

A new minireplicon (rep26 minireplicon) from pBMB26, the 188 kb indigenous plasmid related to spore‐crystal association (SCA) phenotype in Bacillus thuringiensis strain YBT‐020, was characterized. A 12 kb EcoRI fragment, which encoded 10 putative open reading frames (ORFs), was capable of supporting replication when cloned in a replication probe vector. Deletion and frame shift mutation analysis showed that a 4.1 kb region encompassing two putative ORFs (orf21 and orf22) was essential for the plasmid replication in B. thuringiensis. Gene orf21 encoding a 49.8 kDa protein (named Rep26) with a helix‐turn‐helix motif showed no homology with known replication proteins and gene orf22 encoding a protein of 82.6 kDa showed homology to bacterial PcrA helicase. The replication origin of rep26 minireplicon was proved to be located in the coding region of orf21. Plasmid stability experiments indicated that the recombinant plasmid containing rep26 minireplicon has excellent segregational stability. BLASTP analysis revealed that amino acid sequences of ORF21 and ORF22 were well conserved among Bacillus cereus group strains. The rep26 minireplicon was widely distributed and could be defined as a new typical replicon in the megaplasmids of B. cereus group. [ABSTRACT FROM AUTHOR]

Clostridium difficile is a Gram-positive and sporulating enteropathogen that is a major cause of healthcare-associated infections. Even though a large number of genomes of this species have been sequenced, only a few plasmids have been described in the literature. Here, we use a combination of in silico analyses and laboratory experiments to show that plasmids are common in C. difficile . We focus on a group of plasmids that share similarity with the plasmid pCD630, from the reference strain 630. The family of pCD630-like plasmids is defined by the presence of a conserved putative helicase that is likely part of the plasmid replicon. This replicon is compatible with at least some other C. difficile replicons, as strains can carry pCD630-like plasmids in addition to other plasmids. We find two distinct sub-groups of pCD630-like plasmids that differ in size and accessory modules. This study is the first to describe a family of plasmids in C. difficile . [ABSTRACT FROM AUTHOR]

Fluorescence-based methods are widely used to analyze elementary cell processes such as DNA replication or chromosomal folding and segregation. Labeling DNA with a fluorescent protein allows the visualization of its temporal and spatial organization. One popular approach is FROS (fluorescence repressor operator system). This method specifically labels DNA in vivo through binding of a fusion of a fluorescent protein and a repressor protein to an operator array, which contains numerous copies of the repressor binding site integrated into the genomic site of interest. Bound fluorescent proteins are then visible as foci in microscopic analyses and can be distinguished from the background fluorescence caused by unbound fusion proteins. Even though this method is widely used, no attempt has been made so far to decrease the background fluorescence to facilitate analysis of the actual signal of interest. Here, we present a new method that greatly reduces the background signal of FROS. BiFCROS (Bimolecular Fluorescence Complementation and Repressor Operator System) is based on fusions of repressor proteins to halves of a split fluorescent protein. Binding to a hybrid FROS array results in fluorescence signals due to bimolecular fluorescence complementation. Only proteins bound to the hybrid FROS array fluoresce, greatly improving the signal to noise ratio compared to conventional FROS. We present the development of BiFCROS and discuss its potential to be used as a fast and single-cell readout for copy numbers of genetic loci. [ABSTRACT FROM AUTHOR]

• SARS-CoV-2 replicon cDNAs containing HiBiT reporter gene were constructed and cloned into a fosmid vector. • Replication of a series of RNA replicons lacking some open reading frames of SARS-CoV-2 were compared. • Functionalities of SARS-CoV-2 RNA replicons for antiviral drug screening were confirmed. • Replication of SARS-CoV-2 RNA replicons containing puromycin resistant gene showed sustained replication in Huh-7 cells under puromycin pressure. 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 antiviral screening in biosafety level (BSL)-2 facilities. We herein constructed three pCC2Fos-based RNA replicons lacking some open reading frames (ORF) of SARS-CoV-2: the Δorf2–8, Δorf2.4, and Δorf2 replicons, and validated their replication in Huh-7 cells. The functionalities of the Δorf2–8 and Δorf2.4 replicons for antiviral drug screening were also confirmed. We conducted puromycin selection following the construction of the Δorf2.4-puro replicon by inserting a puromycin-resistant gene into the Δorf2.4 replicon. We observed the more sustained replication of the Δorf2.4-puro replicon by puromycin pressure. The present results will contribute to the establishment of a safe and useful replicon system for analyzing SARS-CoV-2 replication mechanisms as well as the development of novel antiviral drugs in BSL-2 facilities. [ABSTRACT FROM AUTHOR]

On the 9th of November 2015, preceding the World Veterinary Vaccine Congress, a workshop was held to discuss how veterinary vaccines can be deployed more rapidly to appropriately respond to future epizootics in Europe. Considering their potential and unprecedented suitability for surge production, the workshop focussed on vaccines based on genetically engineered viruses and replicon particles. The workshop was attended by academics and representatives from leading pharmaceutical companies, regulatory experts, the European Medicines Agency and the European Commission. We here outline the present regulatory pathways for genetically engineered vaccines in Europe and describe the incentive for the organization of the pre-congress workshop. The participants agreed that existing European regulations on the deliberate release of genetically engineered vaccines into the environment should be updated to facilitate quick deployment of these vaccines in emergency situations. [ABSTRACT FROM AUTHOR]

58I.58II.59III.59IV.60V.6263References63 Summary: Viruses are intracellular pathogens that have evolved efficient strategies for replication and expression of their proteins in the host cells. Geminiviruses – plant viruses with small circular single‐stranded DNA genomes – effectively manipulate plant cell processes for viral functions, entailing great potential for biotechnological applications. This potentiality has been realized in the form of protein expression and gene‐silencing vectors, and, more recently, vectors for genome editing – a technology that these viruses seem particularly well‐suited to facilitate. This insight offers an overview of the biological properties of geminiviruses, with emphasis on those leveraging development of geminivirus‐based replicons. It illustrates the basis for engineering geminivirus‐based replicons and their applications. Furthermore, it discusses the reported use and future perspectives of geminivirus‐based replicons for genome editing. Editorial by Lennon & Dolan, 210: 5. [ABSTRACT FROM AUTHOR]

A resident plasmid, pBIF10, was isolated from Bifidobacterium longum B200304, and the full-length sequence of pBIF10 was analyzed. In this sequence, we identified at least 17 major open reading frames longer than 200 bp. A tetracycline resistance gene, tetQ, was identified and verified to confer antibiotic resistance to tetracycline. The plasmid replicon with replication protein B gene ( repB) and a typical iteron was identified in pBIF10. An artificial clone vector was constructed with the replicon of pBIF10; the results showed that repB controlled plasmid replication in other bifidobacteria host cells at low transformation frequency. Taken together, the analysis and characterization of pBIF10 provided necessary information for the understanding of antibiotic resistance mediated by a plasmid in a Bifidobacterium strain. GC% and repB sequence analyses indicated that pBIF10 was a molecular hybrid of at least 2 other bacterial genera plasmids. [ABSTRACT FROM AUTHOR]

Purpose: Foot-and-mouth disease (FMD) is an economically important global animal disease. To control FMD virus (FMDV) outbreaks, a lot of different novel approaches have been attempted. In this study, we proposed a novel porcine reproductive and respiratory syndrome virus (PRRSV) as a replicon vector to express FMDV structural protein. Materials and Methods: PRRSV infectious clone (PRRSVK418DM) was used to develop an expression vector through the reverse genetic manipulation of PRRSV; FMDVP12A3C gene of serotype O was synthesized and used for an antigen. MARC-145 cells (African green monkey kidney epithelial cell line) were used for electroporation mediated transfection. The transfection or the expression of P12A3C and N protein of PRRSV was analyzed by either replicon containing PRRSV alone or by co-infection of helper PRRSV. Results: We constructed PRRSVK418DM replicon vector containing FMDVP12A3C, and genome sequences were confirmed by subsequent sequence analysis. In vitro expression of P12A3C and PRRSV N protein was confirmed by immunofluorescence antibody assay using antibodies specific for PRRSV N protein (anti-PRRSV N MAb), FMDV-VP1 (anti-VP1 MAb). Conclusion: The results indicate that PRRSV replicon vector can be a promising novel vector system to control FMDV and useful for vaccine development in the future. [ABSTRACT FROM AUTHOR]

Highlights: [•] Salmonid alphavirus (SAV) replicon is active in cells from fish, mammals, insects and crustaceans. [•] Salmonid alphavirus replicon can produce gene of interest at temperature range 4–37°C. [•] Fish cells transfected with SAV-replicon plasmid expressed double stranded RNA for several days. [•] Transfection with SAV-replicon gave low toxicity and moderate amount of gene of interest. [ABSTRACT FROM AUTHOR]

Highlights: [•] The pG6302 replicate via theta mechanism and have a novel replicon. [•] pG6303 was thought to replicate via a completely new mechanism. [•] All the orfs of three plasmids were annotated and carried functional analysis. [Copyright &y& Elsevier]

Abstract: Though a modified live attenuated vaccine (MLV) is available against porcine reproductive and respiratory syndrome virus (PRRSV), its limitations in protective efficacy, safety and few others warrant the development of newer vaccines. In this study, we have constructed a propagation-defective DNA-launched PRRSV replicon as a vaccine candidate and evaluated its immunogenicity and protective efficacy in a group of pigs along with MLV vaccinated group. Our data showed that prior to the intranasal challenge with a homologous strain of PRRSV, only MLV vaccinated pigs developed antibody response measured by ELISA and none of the pigs in any group developed PRRSV neutralizing antibodies in serum. The MLV vaccinated group also showed high PRRSV-specific INF-γ response, whereas the replicon-vaccinated pigs showed low but detectable INF-γ response. After 14 days post challenge, all groups showed similar PRRSV-specific serum neutralizing titers and were positive for PRRSV-specific ELISA antibody. In addition, the replicon-vaccinated group showed a significant reduction in viremia in comparison to the control group. In conclusion, vaccination with the PRRSV DNA-launched replicon decreased the viremia and viral load in bronchoalveolar lavage fluids of the PRRSV-challenged pigs and increased numbers of IFN-γ producing cells. Thus, the vaccine is partially protective and is a potential vaccine candidate for future with further improvement. The possible means of improvement is the expression of immunostimulatory genes by the replicon. We demonstrated the feasibility of this approach by expression of a foreign gene encoding firefly luciferase after transfection of cultured cells with the replicon plasmid DNA. [Copyright &y& Elsevier]

Abstract: A replicon-based DNA vaccine encoding VP2 gene of canine parvovirus (CPV) was developed by cloning CPV-VP2 gene into a replicon-based DNA vaccine vector (pAlpha). The characteristics of a replicon-based DNA vaccine like, self-amplification of transcripts and induction of apoptosis were analyzed in transfected mammalian cells. When the pAlpha-CPV-VP2 was injected intradermal as DNA-launched replicon-based DNA vaccine in dogs, it induced CPV-specific humoral and cell mediated immune responses. The virus neutralization antibody and lymphocyte proliferative responses were higher than conventional CPV DNA vaccine and commercial CPV vaccine. These results indicated that DNA-launched replicon-based CPV DNA vaccine was effective in inducing both CPV-specific humoral and cellular immune responses and can be considered as effective alternative to conventional CPV DNA vaccine and commercial CPV vaccine. [Copyright &y& Elsevier]

Abstract: Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was ∼9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections. [Copyright &y& Elsevier]

Abstract: With the exception of nucleoside analogs, few direct acting antivirals in clinical development are active across the six major hepatitis C virus genotypes. We report novel consensus sequence chimeras for genotypes 2b, 3a, 4a, 5a, and 6a NS5B and show variable susceptibilities over a panel of NS5B inhibitors. Tegobuvir (GS-9190) had EC50s of <16nM against genotype 1 and >100nM for other genotypes tested here. An NS5B F445C mutation engineered into the GT3a, 4a, and 6a chimeric replicons lowered the tegobuvir EC50 to levels comparable to those for genotype 1a, but did not considerably alter the EC50 of site 2 or nucleoside analog inhibitors. These data support the use of HCV chimeras in profiling direct acting antivirals across genotypes and specifically determines the impact of the C445F NS5B polymorphism on tegobuvir potency against genotypes 3a, 4a, and 6a. [Copyright &y& Elsevier]

Abstract: Replicon particles derived from Venezuelan equine encephalitis virus (VEE) are infectious non-propagating particles which act as a safe and potent systemic, mucosal, and cellular adjuvant when delivered with antigen. VEE and VEE replicon particles (VRP) can target multiple cell types including dendritic cells (DCs). The role of these cell types in VRP adjuvant activity has not been previously evaluated, and for these studies we focused on the contribution of DCs to the response to VRP. By analysis of VRP targeting in the draining lymph node, we found that VRP induced rapid recruitment of TNF-secreting monocyte-derived inflammatory dendritic cells. VRP preferentially infected these inflammatory DCs as well as classical DCs and macrophages, with less efficient infection of other cell types. DC depletion suggested that the interaction of VRP with classical DCs was required for recruitment of inflammatory DCs, induction of high levels of many cytokines, and for stable transport of VRP to the draining lymph node. Additionally, in vitro-infected DCs enhanced antigen-specific responses by CD4 and CD8 T cells. By transfer of VRP-infected DCs into mice we showed that these DCs generated an inflammatory state in the draining lymph node similar to that achieved by VRP injection. Most importantly, VRP-infected DCs were sufficient to establish robust adjuvant activity in mice comparable to that produced by VRP injection. These findings indicate that VRP infect, recruit and activate both classical and inflammatory DCs, and those DCs become mediators of the VRP adjuvant activity. [Copyright &y& Elsevier]

Abstract: The large pXO1 plasmid (181.6kb) of Bacillus anthracis encodes the anthrax toxin proteins. Previous studies have shown that two separate regions of pXO1 can support replication of pXO1 miniplasmids when introduced into plasmid-less strains of this organism. No information is currently available on the ability of the above two replicons, termed RepX and ORFs 14/16 replicons, to support replication of the full-length pXO1 plasmid. We generated mutants of the full-length pXO1 plasmid in which either the RepX or the ORFs 14/16 replicon was inactivated by TargeTron insertional mutagenesis. Plasmid pXO1 derivatives containing only the RepX or the ORFs 14/16 replicon were able to replicate when introduced into a plasmid-less B. anthracis strain. Plasmid copy number analysis showed that the ORFs 14/16 replicon is more efficient than the RepX replicon. Our studies demonstrate that both the RepX and ORFs 14/16 replicons can independently support the replication of the full-length pXO1 plasmid. [Copyright &y& Elsevier]

Abstract: In this study, a replicon vaccine vector system for Japanese encephalitis virus (JEV) was established. The system included a trans-complementing cell line, a series of JEV DNA-based subgenomic replicons, and several encapsidated JEV propagation-deficient pseudoinfectious particles (PIPs). The DNA-based JEV replicon vectors, which deleted the structural coding region, could be able to self-replicate and express the reporter gene. A stable BHK packaging cell line named BHK-CME, which constitutively expressed the capsid protein C, the precursor membrane and envelope proteins (C-prM-E) of JEV, was generated. BHK-CME cells were used to trans-complement the JEV replicons and proved to package the JEV replicons into single-round infectious PIPs efficiently. The PIPs were produced in titers of up to 1.6×105 IU/ml. To investigate the efficacy of JEV replicon-based vaccines, four groups of female BALB/c mice were inoculated three times at 3-week intervals with the JEV PIPs and others. The JEV-specific antibody titers reached to 1:6400 and the neutralizing antibody titers reached 1:256 after three rounds of immunization with JEV PIPs. And the antisera collected from immunized mice were shown to be protective partially against lethal infection when passively transferred to susceptible weanling mice. These results demonstrated the value of the JEV replicon vector system for the development of new vaccine candidates. [Copyright &y& Elsevier]

The Lactococcus lactis bacterium found in different natural environments is traditionally associated with the fermented food industry. But recently, its applications have been spreading to the pharmaceutical industry, which has exploited its probiotic characteristics and is moving towards its use as cell factories for the production of added-value recombinant proteins and plasmid DNA (pDNA) for DNA vaccination, as a safer and industrially profitable alternative to the traditional Escherichia coli host. Additionally, due to its food-grade and generally recognized safe status, there have been an increasing number of studies about its use in live mucosal vaccination. In this review, we critically systematize the plasmid replicons available for the production of pharmaceutical-grade pDNA and recombinant proteins by L. lactis. A plasmid vector is an easily customized component when the goal is to engineer bacteria in order to produce a heterologous compound in industrially significant amounts, as an alternative to genomic DNA modifications. The additional burden to the cell depends on plasmid copy number and on the expression level, targeting location and type of protein expressed. For live mucosal vaccination applications, besides the presence of the necessary regulatory sequences, it is imperative that cells produce the antigen of interest in sufficient yields. The cell wall anchored antigens had shown more promising results in live mucosal vaccination studies, when compared with intracellular or secreted antigens. On the other side, engineering L. lactis to express membrane proteins, especially if they have a eukaryotic background, increases the overall cellular burden. The different alternative replicons for live mucosal vaccination, using L. lactis as the DNA vaccine carrier or the antigen producer, are critically reviewed, as a starting platform to choose or engineer the best vector for each application. [ABSTRACT FROM AUTHOR]

Background Hospital drains may be a source of CPE in patients. We determined prevalence of and risk factors for CPE contamination of hospital drains exposed to patients with CPE colonization/infection. Methods We cultured hand hygiene and patient use sink as well as tub/shower drains exposed to 310 inpatients colonized/infected with CPE in 10 Ontario hospitals. A multi-level logistic regression model was fitted to determine whether type of drain/room/unit was associated with CPE drain contamination. Drain and room occupant CPE isolates underwent Illumina whole-genome and MinION sequencing. Single nucleotide variant (SNV) phlogenomic analyses and plasmid characterization were performed. Results 53/ Of the 1,209 drains, 53 (4%) at 7 (70%) hospitals yielded CPE. Patient room shower drains were significantly more likely to yield CPE than hand hygiene sink (OR 3.35, 95% CI 1.61–6.97) and patient use sink (OR 10.89, 95% CI 3.62–32.80) drains. Hand hygiene sink drains were significantly more likely to yield CPE than patient use sink drains (OR 3.26, 95% CI 1.05–10.13). 8 (15%) drain isolates matched the room occupant CPE gene/species; 24 (44%) matched the gene but not species, and 23 (42%) matched neither gene nor species. Among 13 drain/11 room occupant pairs with molecular comparisons to date, only 1/13 (8%) drain isolates matched the respective room occupant carbapenemase allele and replicon harboring the carbapenemase gene (IncN replicon harboring bla KPC-3). In addition, one drain isolate had a plasmid highly related to plasmids of 2 isolates from a patient occupying a room in an adjacent unit (IncN3 replicons harboring bla KPC-2). At another hospital, all 6 drain isolates were located on one unit and had an IncHI2A/HI2/TrfA replicon harboring bla NDM-1; 5 of these were E. cloacae ST66, with 0–3 SNV differences observed between isolates. Conclusion Different drain types have different risks of CPE contamination. In our setting, most drain contamination was not caused by recognized room occupants. Further investigation is needed to understand the sources of hospital drain CPE contamination. Disclosures All authors: No reported disclosures. [ABSTRACT FROM AUTHOR]