scholarly journals Efficient and Robust Paramyxoviridae Reverse Genetics Systems

mSphere ◽  
2017 ◽  
Vol 2 (2) ◽  
Author(s):  
Shannon M. Beaty ◽  
Arnold Park ◽  
Sohui T. Won ◽  
Patrick Hong ◽  
Michael Lyons ◽  
...  
Keyword(s):  
Life Cycle ◽  
Reverse Genetics ◽  
High Efficiency ◽  
Hammerhead Ribozyme ◽  
Viral Pathogenesis ◽  
Viral Life Cycle ◽  
Vaccine Vectors ◽  
T7 Promoter ◽  
Transfected Cells ◽  
Low Efficiency

ABSTRACT The ability to manipulate the genome of paramyxoviruses and evaluate the effects of these changes at the phenotypic level is a powerful tool for the investigation of specific aspects of the viral life cycle and viral pathogenesis. However, reverse genetics systems for paramyxoviruses are notoriously inefficient, when successful. The ability to efficiently and robustly rescue paramyxovirus reverse genetics systems can be used to answer basic questions about the biology of paramyxoviruses, as well as to facilitate the considerable translational efforts being devoted to developing live attenuated paramyxovirus vaccine vectors. The notoriously low efficiency of Paramyxoviridae reverse genetics systems has posed a limiting barrier to the study of viruses in this family. Previous approaches to reverse genetics have utilized a wide variety of techniques to overcome the technical hurdles. Although robustness (i.e., the number of attempts that result in successful rescue) has been improved in some systems with the use of stable cell lines, the efficiency of rescue (i.e., the proportion of transfected cells that yield at least one successful rescue event) has remained low. We have substantially increased rescue efficiency for representative viruses from all five major Paramyxoviridae genera (from ~1 in 106-107 to ~1 in 102-103 transfected cells) by the addition of a self-cleaving hammerhead ribozyme (Hh-Rbz) sequence immediately preceding the start of the recombinant viral antigenome and the use of a codon-optimized T7 polymerase (T7opt) gene to drive paramyxovirus rescue. Here, we report a strategy for robust, reliable, and high-efficiency rescue of paramyxovirus reverse genetics systems, featuring several major improvements: (i) a vaccinia virus-free method, (ii) freedom to use any transfectable cell type for viral rescue, (iii) a single-step transfection protocol, and (iv) use of the optimal T7 promoter sequence for high transcription levels from the antigenomic plasmid without incorporation of nontemplated G residues. The robustness of our T7opt-HhRbz system also allows for greater latitude in the ratios of transfected accessory plasmids used that result in successful rescue. Thus, our system may facilitate the rescue and interrogation of the increasing number of emerging paramyxoviruses. IMPORTANCE The ability to manipulate the genome of paramyxoviruses and evaluate the effects of these changes at the phenotypic level is a powerful tool for the investigation of specific aspects of the viral life cycle and viral pathogenesis. However, reverse genetics systems for paramyxoviruses are notoriously inefficient, when successful. The ability to efficiently and robustly rescue paramyxovirus reverse genetics systems can be used to answer basic questions about the biology of paramyxoviruses, as well as to facilitate the considerable translational efforts being devoted to developing live attenuated paramyxovirus vaccine vectors.

scholarly journals Dual Promoters Improve the Rescue of Recombinant Measles Virus in Human Cells

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1723
Author(s):  
Soroth Chey ◽  
Juliane Maria Palmer ◽  
Laura Doerr ◽  
Uwe Gerd Liebert
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Measles Virus ◽  
Reverse Genetics ◽  
Hammerhead Ribozyme ◽  
Polymerase Activity ◽  
T7 Promoter ◽  
Gene Delivery Vectors ◽  
Cloned Cdna ◽  
Dual Promoters

Reverse genetics is a technology that allows the production of a virus from its complementary DNA (cDNA). It is a powerful tool for analyzing viral genes, the development of novel vaccines, and gene delivery vectors. The standard reverse genetics protocols are laborious, time-consuming, and inefficient for negative-strand RNA viruses. A new reverse genetics platform was established, which increases the recovery efficiency of the measles virus (MV) in human 293-3-46 cells. The novel features compared with the standard system involving 293-3-46 cells comprise (a) dual promoters containing the RNA polymerase II promoter (CMV) and the bacteriophage T7 promoter placed in uni-direction on the same plasmid to enhance RNA transcription; (b) three G nucleotides added just after the T7 promoter to increase the T7 RNA polymerase activity; and (c) two ribozymes, the hairpin hammerhead ribozyme (HHRz), and the hepatitis delta virus ribozyme (HDVrz), were used to cleavage the exact termini of the antigenome RNA. Full-length antigenome cDNA of MV of the wild type IC323 strain or the vaccine AIK-C strain was inserted into the plasmid backbone. Both virus strains were easily rescued from their respective cloned cDNA. The rescue efficiency increased up to 80% compared with the use of the standard T7 rescue system. We assume that this system might be helpful in the rescue of other human mononegavirales.

scholarly journals The dimerisable Cre recombinase allows conditional genome editing in the mosquito stages of Plasmodium berghei

2020 ◽  
Author(s):  
Priyanka Fernandes ◽  
Sylvie Briquet ◽  
Delphine Patarot ◽  
Manon Loubens ◽  
Bénédicte Hoareau-Coudert ◽  
...  
Keyword(s):  
Life Cycle ◽  
Dna Sequences ◽  
Malaria Parasite ◽  
Reverse Genetics ◽  
High Efficiency ◽  
Parasite Life Cycle ◽  
Asexual Blood Stage ◽  
Functional Studies ◽  
Powerful Approach ◽  
Regulatable Promoters

ABSTRACTAsexual blood stages of the malaria parasite are readily amenable to genetic modification via homologous recombination, allowing functional studies of parasite genes that are not essential in this part of the life cycle. However, conventional reverse genetics cannot be applied for the functional analysis of genes that are essential during asexual blood-stage replication. Various strategies have been developed for conditional mutagenesis of Plasmodium, including recombinase-based gene deletion, regulatable promoters, and mRNA or protein destabilization systems. Among these, the dimerisable Cre (DiCre) recombinase system has emerged as a powerful approach for conditional gene targeting in P. falciparum. In this system, the bacteriophage Cre is expressed in the form of two separate, enzymatically inactive polypeptides, each fused to a different rapamycin-binding protein. Rapamycin-induced heterodimerization of the two components restores recombinase activity. We have implemented the DiCre system in the rodent malaria parasite P. berghei, and show that rapamycin-induced excision of floxed DNA sequences can be achieved with very high efficiency in both mammalian and mosquito parasite stages. This tool can be used to investigate the function of essential genes not only in asexual blood stages, but also in other parts of the malaria parasite life cycle.

Infection and immunity

2011 ◽  
pp. 797-866
Keyword(s):  
Innate Immunity ◽  
Life Cycle ◽  
Bacterial Growth ◽  
Viral Pathogenesis ◽  
Bacterial Pathogenesis ◽  
Viral Life Cycle ◽  
Viral Therapy ◽  
Bacterial Classification ◽  
Viral Structure ◽  
Bacterial Structure

Pathogens Bacterial structure 798 Bacterial growth 800 Bacterial classification 801 Bacterial pathogenesis 802 Antibiotics 804 Viral structure and classification 808 Viral life cycle 810 Viral pathogenesis 812 Viral infection 813 Viral therapy 814 Prions 816 Fungi 816 Parasites 818 Defence against infection Innate immunity 820...

scholarly journals CHROMOSOMAL BASIS OF THE MEROZYGOSITY IN A PARTIALLY DIPLOID MUTANT OF PNEUMOCOCCUS

Genetics ◽  
1975 ◽  
Vol 80 (4) ◽  
pp. 667-678
Author(s):  
Mary Lee S Ledbetter ◽  
Rollin D Hotchkiss
Keyword(s):  
High Efficiency ◽  
Point Mutations ◽  
Resistant Mutant ◽  
Chromosomal Marker ◽  
Structural Abnormality ◽  
C Cells ◽  
Wild Type ◽  
Chromosomal Locus ◽  
Low Efficiency ◽  
Derivatives Of

ABSTRACT A sulfonamide-resistant mutant of pneumococcus, sulr-c, displays a genetic instability, regularly segregating to wild type. DNA extracts of derivatives of the strain possess transforming activities for both the mutant and wild-type alleles, establishing that the strain is a partial diploid. The linkage of sulr-c to strr-61, a stable chromosomal marker, was established, thus defining a chromosomal locus for sulr-c. DNA isolated from sulr-c cells transforms two mutant recipient strains at the same low efficiency as it does a wild-type recipient, although the mutant property of these strains makes them capable of integrating classical "low-efficiency" donor markers equally as efficiently as "high efficiency" markers. Hence sulr-c must have a different basis for its low efficiency than do classical low efficiency point mutations. We suggest that the DNA in the region of the sulr-c mutation has a structural abnormality which leads both to its frequent segregation during growth and its difficulty in efficiently mediating genetic transformation.

scholarly journals A Brief Review on Solvent-Free Synthesis of Zeolites

Materials ◽  
2021 ◽  
Vol 14 (4) ◽  
pp. 788
Author(s):  
Jinlin Mei ◽  
Aijun Duan ◽  
Xilong Wang
Keyword(s):  
High Efficiency ◽  
Raw Materials ◽  
Solvent Free ◽  
High Yield ◽  
Control Synthesis ◽  
Mechanical Mixing ◽  
Synthesis Mechanism ◽  
Wastewater Pollution ◽  
Low Efficiency ◽  
Solvent Free Synthesis

The traditional hydrothermal method to prepare zeolite will inevitably use a large amount of water as a solvent, which will lead to higher autogenous pressure, low efficiency, and wastewater pollution. The solvent-free method can be used to synthesize various types of zeolites by mechanical mixing, grinding, and heating of solid raw materials, which exhibits the apparent advantages of high yield, low pollution, and high efficiency. This review mainly introduces the development process of solvent-free synthesis, preparation of hierarchical zeolite, morphology control, synthesis mechanism and applications of solvent-free methods. It can be believed that solvent-free methods will become a research focus and have enormous industrial application potential.

scholarly journals Nucleosome Positioning on Episomal Human Papillomavirus DNA in Cultured Cells

Pathogens ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 772
Author(s):  
Isao Murakami ◽  
Takashi Iwata ◽  
Tohru Morisada ◽  
Kyoko Tanaka ◽  
Daisuke Aoki
Keyword(s):  
Life Cycle ◽  
Cultured Cells ◽  
Basal Layer ◽  
Nucleosome Positioning ◽  
Viral Life Cycle ◽  
Human Papillomaviruses ◽  
Host Cells ◽  
Micrococcal Nuclease ◽  
Hpv Dna ◽  
Dna Replication And Repair

Several human papillomaviruses (HPV) are associated with the development of cervical carcinoma. HPV DNA synthesis is increased during the differentiation of infected host keratinocytes as they migrate from the basal layer of the epithelium to the spinous layer, but the molecular mechanism is unclear. Nucleosome positioning affects various cellular processes such as DNA replication and repair by permitting the access of transcription factors to promoters to initiate transcription. In this study, nucleosome positioning on virus chromatin was investigated in normal immortalized keratinocytes (NIKS) stably transfected with HPV16 or HPV18 genomes to determine if there is an association with the viral life cycle. Micrococcal nuclease-treated DNA analyzed by Southern blotting using probes against HPV16 and HPV18 and quantified by nucleosome scanning analysis using real-time PCR revealed mononucleosomal-sized fragments of 140–200 base pairs that varied in their location within the viral genome according to whether the cells were undergoing proliferation or differentiation. Notably, changes in the regions around nucleotide 110 in proliferating and differentiating host cells were common to HPV16 and HPV18. Our findings suggest that changes in nucleosome positions on viral DNA during host cell differentiation is an important regulatory event in the viral life cycle.

scholarly journals Inflight Transmission of COVID-19 Based on Experimental Aerosol Dispersion Data

2021 ◽  
Author(s):  
Zhaozhi Wang ◽  
Edwin R Galea ◽  
Angus Grandison ◽  
John Ewer ◽  
Fuchen Jia
Keyword(s):  
High Efficiency ◽  
Face Mask ◽  
Generation Rate ◽  
Aviation Industry ◽  
Filtration System ◽  
Dispersion Data ◽  
Low Probability ◽  
Meal Service ◽  
Low Efficiency ◽  
Aerosol Dispersion

Abstract Background An issue of concern to the travelling public is the possibility of in-flight transmission of COVID-19 during long- and short-haul flights. The aviation industry maintains that the probability of contracting the illness is small based on reported cases, modelling and data from aerosol dispersion experiments conducted on-board aircraft. Methods Using experimentally derived aerosol dispersion data for a B777–200 aircraft and a modified version of the Wells-Riley equation we estimate inflight infection probability for a range of scenarios involving quanta generation rate and face mask efficiency. Quanta generation rates were selected based on COVID-19 events reported in the literature while mask efficiency was determined from the aerosol dispersion experiments. Results The MID-AFT cabin exhibits the highest infection probability. The calculated maximum individual infection probability (without masks) for a 2-hour flight in this section varies from 4.5% for the ‘Mild Scenario’ to 60.2% for the ‘Severe Scenario’ although the corresponding average infection probability varies from 0.1% to 2.5%. For a 12-hour flight, the corresponding maximum individual infection probability varies from 24.1% to 99.6% and the average infection probability varies from 0.8% to 10.8%. If all passengers wear face masks throughout the 12-hour flight, the average infection probability can be reduced by approximately 73%/32% for high/low efficiency masks. If face masks are worn by all passengers except during a one-hour meal service, the average infection probability is increased by 59%/8% compared to the situation where the mask is not removed. Conclusions This analysis has demonstrated that while there is a significant reduction in aerosol concentration due to the nature of the cabin ventilation and filtration system, this does not necessarily mean that there is a low probability or risk of in-flight infection. However, mask wearing, particularly high-efficiency ones, significantly reduces this risk.

scholarly journals Sustainable Ventilation Strategies for a Medium-Sized Space with Regional Effect

2021 ◽  
Vol 13 (9) ◽  
pp. 4651
Author(s):  
Ming-Lun Alan Fong
Keyword(s):  
Life Cycle ◽  
Life Cycle Cost ◽  
Co2 Emission ◽  
High Efficiency ◽  
Regional Climate ◽  
Cost Effective ◽  
Regional Effect ◽  
Climate Conditions ◽  
Ventilation Strategies ◽  
Set Up

The analysis of ventilation strategies is fundamentally affected by regional climate conditions and local cost databases, in terms of energy consumption, CO2 emission and cost-effective analysis. A systematic approach is covered in this paper to estimate a local economic and environmental impact on a medium-sized space located in two regions during supply-and-installation and operation phases. Three ventilation strategies, including mixing ventilation (MV), displacement ventilation (DV) and stratum ventilation (SV) were applied to medium-sized air-conditioned space with this approach. The trend of the results for three ventilation systems in the life cycle assessment (LCA) and life cycle cost (LCC) analysis is SV < DV < MV. The result of CO2 emission and regional LCC shows that SV is the lowest one in both regional studies. In comparison with the Hong Kong Special Administrative Region (HKSAR) during 20 Service years, the case analysis demonstrates that the percentage differences in LCC analysis of MV, DV & SV in Guangdong are less than 20.5%, 19.4% and 18.82% respectively. Their CO2 emission of MV, DV and SV in Guangdong are more than HKSAR in 10.69%, 11.22% and 12.05%, respectively. The present study could provide information about regional effects in the LCA and LCC analysis of three ventilation strategies emissions, and thereby help set up models for decision-making on high efficiency and cost-effective ventilation strategy plans.

scholarly journals A Comprehensive, Flexible Collection of SARS-CoV-2 Coding Regions

2020 ◽  
Vol 10 (9) ◽  
pp. 3399-3402 ◽  
Author(s):  
Dae-Kyum Kim ◽  
Jennifer J Knapp ◽  
Da Kuang ◽  
Aditya Chawla ◽  
Patricia Cassonnet ◽  
...  
Keyword(s):  
Life Cycle ◽  
Viral Life Cycle ◽  
Coding Sequences ◽  
Coding Regions ◽  
Global Pandemic ◽  
The World ◽  
Molecular Studies ◽  
Widespread Availability ◽  
Rapid Transfer

Abstract The world is facing a global pandemic of COVID-19 caused by the SARS-CoV-2 coronavirus. Here we describe a collection of codon-optimized coding sequences for SARS-CoV-2 cloned into Gateway-compatible entry vectors, which enable rapid transfer into a variety of expression and tagging vectors. The collection is freely available. We hope that widespread availability of this SARS-CoV-2 resource will enable many subsequent molecular studies to better understand the viral life cycle and how to block it.

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