Semliki Forest Virus (SFV) Self-Amplifying RNA Delivered to J774A.1 Macrophage Lineage by Its Association with a Purified Recombinant SFV Capsid Protein.

The Semliki Forest virus capsid protein (C) is an RNA binding protein which exhibits both specific and unspecific affinities to single-strand nucleic acids. The putative use of the self-amplifying RNAs (saRNAs) of alphaviruses for biotechnological purpose is one of the main studied strategies concerning RNA-based therapies or immunization. In this work, a recombinant C protein from SFV was expressed and purified from bacteria and used to associate in vitro with a saRNA derived from SFV. Results showed that the purified form of C protein can associate with the saRNA even after high temperature treatment. The C protein was associated with a modified saRNA coding for the green fluorescent protein (GFP) and delivered to murine macrophage cells which expressed the GFP, showing that the saRNA was functional after being associated with the recombinant purified C protein.

RNA Combined with Nanoformulation to Advance Therapeutic Technologies.

Nucleic acid-based therapies have the potential to address numerous diseases that pose significant challenges to more traditional methods. RNA-based therapies have emerged as a promising avenue, utilizing nanoformulation treatments to target a range of pathologies. Nanoformulation offers several advantages compared to other treatment modalities, including targeted delivery, low toxicity, and bioactivity suitable for drug loading. At present, various types of nanoformulations are available, such as liposomes, polymeric nanoparticles (NPs), magnetic NPs, nanoshells, and solid lipid nanoparticles (SLNs). RNA-based therapy utilizes intracellular gene nanoparticles with messenger RNA (mRNA) emerging prominently in cancer therapy and immunotechnology against infectious diseases. The approval of mRNA-based technology opens doors for future technological advancements, particularly self-amplifying replicon RNA (repRNA). RepRNA is a novel platform in gene therapy, comprising viral RNA with a unique molecular property that enables the amplification of all encoded genetic information countless times. As a result, repRNA-based therapies have achieved significant levels of gene expression. In this context, the primary objective of this study is to furnish a comprehensive review of repRNA and its applications in nanoformulation treatments, with a specific focus on encapsulated nanoparticles. The overarching goal is to provide an extensive overview of the use of repRNA in conjunction with nanoformulations across a range of treatments and therapies.

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 analysis of the immune response to the WEVEE VRP vaccine. NHPs were challenged by aerosol 28 or 29 days following second vaccination with WEEV CBA87. Vaccination with two doses of WEVEE VRP was immunogenic and resulted in neutralizing antibody responses specific for VEEV, EEEV and WEEV. None of the vaccinated animals met euthanasia criteria following aerosol exposure to WEEV CBA87. However, one NHP control (total of 11 controls) met euthanasia criteria after infection with WEEV CBA87. Statistically significant differences in median fever hours were noted in control NHPs compared to vaccinated NHPs, providing a quantitative measure of infection and efficacy of the vaccine against a WEEV challenge. Alterations in lymphocytes, monocytes, and neutrophils were observed. Lymphopenia was observed in control NHPs.

The Antifungal Itraconazole Is a Potent Inhibitor of Chikungunya Virus Replication.

Chikungunya virus (CHIKV) is the causative agent of chikungunya fever, a disabling disease that can cause long-term severe arthritis. Since the last large CHIKV outbreak in 2015, the reemergence of the virus represents a serious public health concern. The morbidity associated with viral infection emphasizes the need for the development of specific anti-CHIKV drugs. Herein, we describe the development and characterization of a CHIKV reporter replicon cell line and its use in replicon-based screenings. We tested 960 compounds from MMV/DNDi Open Box libraries and identified four candidates with interesting antiviral activities, which were confirmed in viral infection assays employing CHIKV-nanoluc and BHK-21 cells. The most noteworthy compound identified was itraconazole (ITZ), an orally available, safe, and cheap antifungal, that showed high selectivity indexes of >312 and >294 in both replicon-based and viral infection assays, respectively. The antiviral activity of this molecule has been described against positive-sense single stranded RNA viruses (+ssRNA) and was related to cholesterol metabolism that could affect the formation of the replication organelles. Although its precise mechanism of action against CHIKV still needs to be elucidated, our results demonstrate that ITZ is a potent inhibitor of the viral replication that could be repurposed as a broad-spectrum antiviral.

Construction of a Chikungunya Virus, Replicon, and Helper Plasmids for Transfection of Mammalian Cells.

The genome of Alphaviruses can be modified to produce self-replicating RNAs and virus-like particles, which are useful virological tools. In this work, we generated three plasmids for the transfection of mammalian cells: an infectious clone of Chikungunya virus (CHIKV), one that codes for the structural proteins (helper plasmid), and another one that codes nonstructural proteins (replicon plasmid). All of these plasmids contain a reporter gene (mKate2). The reporter gene in the replicon RNA and the infectious clone are synthesized from subgenomic RNA. Co-transfection with the helper and replicon plasmids has biotechnological/biomedical applications because they allow for the delivery of self-replicating RNA for the transient expression of one or more genes to the target cells.

Reporter Replicons for Antiviral Drug Discovery against Positive Single-Stranded RNA Viruses.

Single-stranded positive RNA ((+) ssRNA) viruses include several important human pathogens. Some members are responsible for large outbreaks, such as Zika virus, West Nile virus, SARS-CoV, and SARS-CoV-2, while others are endemic, causing an enormous global health burden. Since vaccines or specific treatments are not available for most viral infections, the discovery of direct-acting antivirals (DAA) is an urgent need. Still, the low-throughput nature of and biosafety concerns related to traditional antiviral assays hinders the discovery of new inhibitors. With the advances of reverse genetics, reporter replicon systems have become an alternative tool for the screening of DAAs. Herein, we review decades of the use of (+) ssRNA viruses replicon systems for the discovery of antiviral agents. We summarize different strategies used to develop those systems, as well as highlight some of the most promising inhibitors identified by the method. Despite the genetic alterations introduced, reporter replicons have been shown to be reliable systems for screening and identification of viral replication inhibitors and, therefore, an important tool for the discovery of new DAAs.

Semliki Forest Virus replicon particles production in serum-free medium BHK-21 cell cultures and their use to express different proteins.

The production of biopharmaceuticals as vaccines in serum-free media results in reduced risk of contamination and simpler downstream processing. The production of enveloped viruses and viral vectors such as Semliki Forest Virus (SFV) typically requires lipids that are provided by supplementation with animal serum, so production under serum-free conditions is challenging. In this work, the capacity to deliver genetic material of SFV-viral replicon particles (SFV-VRPs) produced in BHK-21 cells adapted to serum-free medium (BHK/SFM) was evaluated. Three transgenes were evaluated: GFP used as a model protein, while hepatitis C virus nonstructural protein 3 protease domain (HCV-NS3p) and rabies virus glycoprotein (RVGP) were selected based on their distinct nature (enzyme and glycoprotein, respectively). BHK/SFM cells produced a sevenfold higher number of SFV-VRPs, as determined by qRT-PCR. These particles showed similar capacities of infecting BHK/FBS or BHK/SFM cells. GFP expression was evaluated by flow cytometry, HCV-NS3p activity by enzymatic assay, and RVGP expression by ELISA and Western Blot. Expression analysis revealed higher levels of GFP and HCV-NS3p in BHK/SFM, while the levels of RVGP were similar for BHK/SFM and BHK/FBS. In conclusion, the BHK/SFM cells showed increased SFV-VRP production yields, without affecting vector infectivity or heterologous gene expression, hence validating the use of BHK/SFM for industrial applications.

Design of a microaerobically inducible replicon for high-yield plasmid DNA production.

A pUC-derived replicon inducible by oxygen limitation was designed and tested in fed-batch cultures of Escherichia coli. It included the addition of a second inducible copy of rnaII, the positive replication control element. The rnaII gene was expressed from Ptrc and cloned into pUC18 to test the hypothesis that the ratio of the positive control molecule RNAII to the negative control element, RNAI, was the determinant of plasmid copy number per chromosome (PCN). The construct was evaluated in several E. coli strains. Evaluations of the RNAII/RNAI ratio, PCN and plasmid yield normalized to biomass (YpDNA/X ) were performed and the initial hypothesis was probed. Furthermore, in high cell-density cultures in shake flasks, an outstanding amount of 126 mg/L of plasmid was produced. The microaerobically inducible plasmid was obtained by cloning the rnaII gene under the control of the oxygen-responsive Vitreoscilla stercoraria hemoglobin promoter. For this plasmid, but not for pUC18, the RNAII/RNAI ratio, PCN and YpDNA/X efficiently increased after the shift to the microaerobic regime in fed-batch cultures in a 1 L bioreactor. The YpDNA/X of the inducible plasmid reached 12 mg/g at the end of the fed-batch but the original pUC18 only reached ca. 6 mg/g. The proposed plasmid is a valuable alternative for the operation and scale-up of plasmid DNA production processes in which mass transfer limitations will not represent an issue.

Design of a synthetic miniR1 plasmid and its production by engineered Escherichia coli.

A synthetic plasmid consisting of the minimal elements for replication control of the R1 replicon and kanamycin resistance marker, which was named pminiR1, was developed. pminiR1 production was tested at 30 °C under aerobic and microaerobic conditions in Escherichia coli W3110 recA- (W1). The plasmid DNA yields from biomass (YpDNA/X) were only 0.06 ± 0.02 and 0.22 ± 0.11 mg/g under aerobic and microaerobic conditions, respectively. As an option to increase YpDNA/X values, pminiR1 was introduced in an engineered E. coli strain expressing the Vitreoscilla hemoglobin inserted in chromosome (W12). The YpDNA/X values using strain W12 increased to 0.85 ± 0.05 and 1.53 ± 0.14 mg/g under aerobic and microaerobic conditions, respectively. pminiR1 production in both strains was compared with that of pUC57Kan at 37 °C under aerobic and microaerobic conditions. The YpDNA/X values for pminiR1 using strain W12 were 6.25 ± 0.16 and 9.27 ± 0.95 mg/g under aerobic and microaerobic conditions, respectively. Such yields were similar to those obtained for plasmid pUC57Kan using strain W12 (6.9 ± 0.64 and 10.85 ± 1.06 mg/g for aerobic and microaerobic cultures, respectively). Therefore, the synthetic minimal plasmid based on the R1 replicon is a valuable alternative to pUC plasmids for biotechnological applications.

Synthesis of New Indanyl Nucleoside Analogues and their Biological Evaluation on Hepatitis C Virus (HCV) Replicon.

Here, we report a convenient synthetic procedure for the preparation of four novel indanyl carbanucleoside derivatives in the racemic form. The action of these compounds against hepatitis C virus was evaluated in vitro using the replicon cell line, Huh7.5 SG. Contrary to our expectations, all these compounds did not inhibit, but rather promoted HCV genotype 1b (HCVg1b) replication. Similar effects have been reported for morphine in the replicon cell lines, Huh7 and Huh8. Several biological experiments and computational studies were performed to elucidate the effect of these compounds on HCVg1b replication. Based on all the experiments performed, we propose that the increase in HCVg1b replication could be mediated, at least in part, by a similar mechanism to that of morphine on the enhancement of this replication. The presence of opioid receptors in Huh7.5 SG cells was indirectly determined for the first time in this work.

Semliki Forest Virus replicon particles production in serum-free medium BHK-21 cell cultures and their use to express different proteins

The production of biopharmaceuticals as vaccines in serum-free media results in reduced risk of contamination and simpler downstream processing. The production of enveloped viruses and viral vectors such as Semliki Forest Virus (SFV) typically requires lipids that are provided by supplementation with animal serum, so production under serum-free conditions is challenging. In this work, the capacity to deliver genetic material of SFV-viral replicon particles (SFV-VRPs) produced in BHK-21 cells adapted to serum-free medium (BHK/SFM) was evaluated. Three transgenes were evaluated: GFP used as a model protein, while hepatitis C virus nonstructural protein 3 protease domain (HCV-NS3p) and rabies virus glycoprotein (RVGP) were selected based on their distinct nature (enzyme and glycoprotein, respectively). BHK/SFM cells produced a sevenfold higher number of SFV-VRPs, as determined by qRT-PCR. These particles showed similar capacities of infecting BHK/FBS or BHK/SFM cells. GFP expression was evaluated by flow cytometry, HCV-NS3p activity by enzymatic assay, and RVGP expression by ELISA and Western Blot. Expression analysis revealed higher levels of GFP and HCV-NS3p in BHK/SFM, while the levels of RVGP were similar for BHK/SFM and BHK/FBS. In conclusion, the BHK/SFM cells showed increased SFV-VRP production yields, without affecting vector infectivity or heterologous gene expression, hence validating the use of BHK/SFM for industrial applications.

Development of a Broadly Accessible Venezuelan Equine Encephalitis Virus Replicon Particle Vaccine Platform.

Zoonotic viruses circulate as swarms in animal reservoirs and can emerge into human populations, causing epidemics that adversely affect public health. Portable, safe, and effective vaccine platforms are needed in the context of these outbreak and emergence situations. In this work, we report the generation and characterization of an alphavirus replicon vaccine platform based on a non-select agent, attenuated Venezuelan equine encephalitis (VEE) virus vaccine, strain 3526 (VRP 3526). Using both noroviruses and coronaviruses as model systems, we demonstrate the utility of the VRP 3526 platform in the generation of recombinant proteins, production of virus-like particles, and in vivo efficacy as a vaccine against emergent viruses. Importantly, packaging under biosafety level 2 (BSL2) conditions distinguishes VRP 3526 from previously reported alphavirus platforms and makes this approach accessible to the majority of laboratories around the world. In addition, improved outcomes in the vulnerable aged models as well as against heterologous challenge suggest improved efficacy compared to that of previously attenuated VRP approaches. Taking these results together, the VRP 3526 platform represents a safe and highly portable system that can be rapidly deployed under BSL2 conditions for generation of candidate vaccines against emerging microbial pathogens.IMPORTANCE While VEE virus replicon particles provide a robust, established platform for antigen expression and vaccination, its utility has been limited by the requirement for high-containment-level facilities for production and packaging. In this work, we utilize an attenuated vaccine strain capable of use at lower biocontainment level but retaining the capacity of the wild-type replicon particle. Importantly, the new replicon platform provides equal protection for aged mice and following heterologous challenge, which distinguishes it from other attenuated replicon platforms. Together, the new system represents a highly portable, safe system for use in the context of disease emergence.

Reverse genetic system, genetically stable reporter viruses and packaged subgenomic replicon based on a Brazilian Zika virus isolate.

Zika virus (ZIKV, genus Flavivirus) has emerged as a major mosquito-transmitted human pathogen, with recent outbreaks associated with an increased incidence of neurological complications, particularly microcephaly and the Guillain-Barré syndrome. Because the virus has only very recently emerged as an important pathogen, research is being hampered by a lack of reliable molecular tools. Here we report an infectious cDNA (icDNA) clone for ZIKV isolate BeH819015 from Brazil, which was selected as representative of South American ZIKV isolated at early stages of the outbreak. icDNA clones were assembled from synthetic DNA fragments corresponding to the consensus sequence of the BeH819015 isolate. Virus rescued from the icDNA clone had properties identical to a natural ZIKV isolate from South America. Variants of the clone-derived virus, expressing nanoluciferase, enhanced green fluorescent or mCherry marker proteins in both mammalian and insect cells and being genetically stable for multiple in vitro passages, were obtained. A ZIKV subgenomic replicon, lacking a prM- and E glycoprotein encoding region and expressing a Gaussia luciferase marker, was constructed and shown to replicate both in mammalian and insect cells. In the presence of the Semliki Forest virus replicon, expressing ZIKV structural proteins, the ZIKV replicon was packaged into virus-replicon particles. Efficient reverse genetic systems, genetically stable marker viruses and packaged replicons offer significant improvements for biological studies of ZIKV infection and disease, as well as for the development of antiviral approaches.

Antimicrobial resistance and plasmid replicons in Yersinia enterocolitica strains isolated in Brazil in 30 years

Abstract Some studies evaluated the resistance profile of the Y. enterocolitica strains isolated in diverse countries. However, in Brazil the isolation and the study of Y. enterocolitica are not common and therefore information about the antimicrobial resistance profile of this species in this country is scarce. Therefore, the aim of this study was to evaluate the antimicrobial resistance of Y. enterocolitica of biotypes 1A, 2 and 4 isolated from clinical and non-clinical sources between 1979 and 2012, in Brazil. This study showed that some Yersinia enterocolitica of different biotypes remain susceptible to antimicrobials used for gastroenteritis treatment. Moreover, neither acquired resistance genes nor diversity of plasmids replicons were found; however, variation in the in vitro intrinsic resistant pattern was detected, except the non-resistance to cefoxitin in all strains. Notwithstanding, due to epidemiological link between Y. enterocolitica and the pork production chain, monitoring plasmid acquired resistance in Y. enterocolitica could also be considered for antimicrobial resistance control purposes and food safety measures.

Algevir: An Expression System for Microalgae Based on Viral Vectors.

The use of recombinant algae for the production of valuable compounds is opening promising biotechnological applications. However, the development of efficient expression approaches is still needed to expand the exploitation of microalgae in biotechnology. Herein, the concept of using viral expression vectors in microalgae was explored for the first time. An inducible geminiviral vector leading to Rep-mediated replication of the expression cassette allowed the production of antigenic proteins at high levels. This system, called Algevir, allows the production of complex viral proteins (GP1 from Zaire ebolavirus) and bacterial toxin subunits (B subunit of the heat-labile Escherichia coli enterotoxin), which retained their antigenic activity. The highest achieved yield was 1.25 mg/g fresh biomass (6 mg/L of culture), which was attained 3 days after transformation. The Algevir system allows for a fast and efficient production of recombinant proteins, overcoming the difficulties imposed by the low yields and unstable expression patterns frequently observed in stably transformed microalgae at the nuclear level; as well as the toxicity of some target proteins.

Antimicrobial resistance and plasmid replicons in Yersinia enterocolitica strains isolated in Brazil in 30 years.

Some studies evaluated the resistance profile of the Y. enterocolitica strains isolated in diverse countries. However, in Brazil the isolation and the study of Y. enterocolitica are not common and therefore information about the antimicrobial resistance profile of this species in this country is scarce. Therefore, the aim of this study was to evaluate the antimicrobial resistance of Y. enterocolitica of biotypes 1A, 2 and 4 isolated from clinical and non-clinical sources between 1979 and 2012, in Brazil. This study showed that some Yersinia enterocolitica of different biotypes remain susceptible to antimicrobials used for gastroenteritis treatment. Moreover, neither acquired resistance genes nor diversity of plasmids replicons were found; however, variation in the in vitro intrinsic resistant pattern was detected, except the non-resistance to cefoxitin in all strains. Notwithstanding, due to epidemiological link between Y. enterocolitica and the pork production chain, monitoring plasmid acquired resistance in Y. enterocolitica could also be considered for antimicrobial resistance control purposes and food safety measures.

Generation of a DNA-Launched Reporter Replicon Based on Dengue Virus Type 2 as a Multipurpose Platform.

Dengue viruses (DENV) have become the most important arthropod-borne viruses, causing dengue and severe dengue fever in at least 50-100 million cases each year, mainly in tropical and subtropical countries. During recent years, important advances in the molecular biology concerning the life cycle of these viruses have allowed the manipulation and generation of recombinant viruses and replicons with multiple applications, mainly in viral biology and the screening of antiviral compounds. In the present study, we describe the construction of an enhanced green fluorescent protein-bearing DENV replicon under the control of the cytomegalovirus immediate early promoter. Following a rational in silico design and cloning by standard molecular biology techniques, a reporter DENV-2 replicon and a replication-deficient mutant were constructed, and characterized by confocal microscopy and real-time RT-PCR. The results showed successful transcription, translation, and autonomous viral RNA replication of the DENV replicon from its DNA clone. This novel DENV replicon will allow the study of viral replication and testing of antiviral candidates without the need for in vitro transcription.

Vaccines for viral and bacterial pathogens causing acute gastroenteritis: Part I: Overview, vaccines for enteric viruses and Vibrio cholerae.

Efforts to develop vaccines for prevention of acute diarrhea have been going on for more than 40 y with partial success. The myriad of pathogens, more than 20, that have been identified as a cause of acute diarrhea throughout the years pose a significant challenge for selecting and further developing the most relevant vaccine candidates. Based on pathogen distribution as identified in epidemiological studies performed mostly in low-resource countries, rotavirus, Cryptosporidium, Shigella, diarrheogenic E. coli and V. cholerae are predominant, and thus the main targets for vaccine development and implementation. Vaccination against norovirus is most relevant in middle/high-income countries and possibly in resource-deprived countries, pending a more precise characterization of disease impact. Only a few licensed vaccines are currently available, of which rotavirus vaccines have been the most outstanding in demonstrating a significant impact in a short time period. This is a comprehensive review, divided into 2 articles, of nearly 50 vaccine candidates against the most relevant viral and bacterial pathogens that cause acute gastroenteritis. In order to facilitate reading, sections for each pathogen are organized as follows: i) a discussion of the main epidemiological and pathogenic features; and ii) a discussion of vaccines based on their stage of development, moving from current licensed vaccines to vaccines in advanced stage of development (in phase IIb or III trials) to vaccines in early stages of clinical development (in phase I/II) or preclinical development in animal models. In this first article we discuss rotavirus, norovirus and Vibrio cholerae. In the following article we will discuss Shigella, Salmonella (non-typhoidal), diarrheogenic E. coli (enterotoxigenic and enterohemorragic), and Campylobacter jejuni.

Construction of a cassette for cloning and analysis of replicons / Construccion de un cassette para el clonamiento y analisis de replicones

The aim of this work was the construction of a cassette, i.e., a non-replicative molecule formed by linkage of an antibiotic resistance gene and a multiple cloning site. This cassette would allow the cloning and analysis of a wide range of replicons. The aac(6')-lc amikacin gene was isolated and ligated to the multiple clining site of the pUC18 vector. This construction was HindIII digested and cloned in the HindIII site of the vector. The resulting pHJ13 clone conferred to the recipient cells the ability to grow in presence of amikacin (cassette marker) and ampicillin (vector gene). By restriction analysis, the cassette orientation was established. Cassette versatility is provided by the presence of the unaltered multiple cloning site segment, and also because it allows sequencing of any replication origin inserted. Cassette funcionality was demonstrated by ligation to a replicative region of H plasmid pHH1457. Presence of the ori region from pHH1457 and the aac(6')-lc gene was confirmed in E. coli transformed clones. The incompatibility properties of the pHH1457 and its capability to replicate in a Poll defective strain were preserved in the pHJII14 construct. Currently, the amikacin cassette is being used in the characterization of H Complex plasmids.

El objetivo de este trabajo es la construcción de un cassette ­ molécula no replicativa ­ formada por un gen de resistencia a un antibiótico y una región de múltiple sitios de clonamiento. Este cassette permitirá el clonamiento y análisis de una amplia variedad de replicones. El gen que determina resistencia a amikacina (aac (6')-Ic) fue aislado y ligado a la región de múltiple sitios de clonamiento del vector pUC18. La construcción resultante fue digerida con Hind III y clonada en el sitio Hind III del vector. El clon pHJ13 resultante confirió a las células receptoras la capacidad de crecer en presencia de amikacina (marcador del cassette) y ampicilina (marcador del vector). Mediante análisis con enzimas de restricción se determinó la orientación del cassette. La versatilidad del cassette se sustenta en la presencia, sin modificaciones, de la región de múltiple sitios de clonamiento, que permitirá obtener la secuencia de nucleótidos de cualquier origen de replicación insertado. La funcionalidad del cassette fue demostrada mediante el ligamiento a una región de replicación del plásmido pHH1457 (Complejo H). La presencia de la región ori de pHH1457 y del gen aac (6')-Ic fue confirmada en clones de E. coli. Las propiedades de incompatibilidad del plásmido H y su capacidad para replicarse en una cepa defectiva en PolI se conservaron en el plásmido pHJII14 construido. El cassette de amikacina está siendo utilizado en la caracterización de plásmidos del Complejo H. P