scholarly journals Recovery of genetically defined murine norovirus in tissue culture by using a fowlpox virus expressing T7 RNA polymerase

2007 ◽  
Vol 88 (8) ◽  
pp. 2091-2100 ◽  
Author(s):  
Yasmin Chaudhry ◽  
Michael A. Skinner ◽  
Ian G. Goodfellow
Keyword(s):  
Tissue Culture ◽  
Rna Polymerase ◽  
Reverse Genetics ◽  
Low Cost ◽  
T7 Rna Polymerase ◽  
Murine Norovirus ◽  
Fowlpox Virus ◽  
Negative Effect ◽  
Virus Recovery ◽  
First Time

Despite the significant disease burden caused by human norovirus infection, an efficient tissue-culture system for these viruses remains elusive. Murine norovirus (MNV) is an ideal surrogate for the study of norovirus biology, as the virus replicates efficiently in tissue culture and a low-cost animal model is readily available. In this report, a reverse-genetics system for MNV is described, using a fowlpox virus (FWPV) recombinant expressing T7 RNA polymerase to recover genetically defined MNV in tissue culture for the first time. These studies demonstrated that approaches that have proved successful for other members of the family Caliciviridae failed to lead to recovery of MNV. This was due to our observation that vaccinia virus infection had a negative effect on MNV replication. In contrast, FWPV infection had no deleterious effect and allowed the recovery of infectious MNV from cells previously transfected with MNV cDNA constructs. These studies also indicated that the nature of the 3′-terminal nucleotide is critical for efficient virus recovery and that inclusion of a hepatitis delta virus ribozyme at the 3′ end can increase the efficiency with which virus is recovered. This system now allows the recovery of genetically defined noroviruses and will facilitate the analysis of the effects of genetic variation on norovirus pathogenesis.

scholarly journals Expression of bacteriophage T7 RNA polymerase in avian and mammalian cells by a recombinant fowlpox virus

1996 ◽  
Vol 77 (5) ◽  
pp. 963-967 ◽  
Author(s):  
P. Britton ◽  
P. Green ◽  
S. Kottier ◽  
K. L. Mawditt ◽  
Z. Penzes ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Mammalian Cells ◽  
T7 Rna Polymerase ◽  
Bacteriophage T7 ◽  
Fowlpox Virus ◽  
Recombinant Fowlpox Virus

scholarly journals Selection and Characterization of Rupintrivir-Resistant Norwalk Virus Replicon Cells In Vitro

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
Mitsutaka Kitano ◽  
Myra Hosmillo ◽  
Edward Emmott ◽  
Jia Lu ◽  
Ian Goodfellow
Keyword(s):  
Reverse Genetics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Murine Norovirus ◽  
Norwalk Virus ◽  
Irreversible Inhibitor ◽  
Antiviral Compounds ◽  
A Cell ◽  
First Time

ABSTRACT Human norovirus (HuNoV) is a major cause of nonbacterial gastroenteritis worldwide, yet despite its impact on society, vaccines and antivirals are currently lacking. A HuNoV replicon system has been widely applied to the evaluation of antiviral compounds and has thus accelerated the process of drug discovery against HuNoV infection. Rupintrivir, an irreversible inhibitor of the human rhinovirus 3C protease, has been reported to inhibit the replication of the Norwalk virus replicon via the inhibition of the norovirus protease. Here we report, for the first time, the generation of rupintrivir-resistant human Norwalk virus replicon cells in vitro . Sequence analysis revealed that these replicon cells contained amino acid substitutions of alanine 105 to valine (A105V) and isoleucine 109 to valine (I109V) in the viral protease NS6. The application of a cell-based fluorescence resonance energy transfer (FRET) assay for protease activity demonstrated that these substitutions were involved in the enhanced resistance to rupintrivir. Furthermore, we validated the effect of these mutations using reverse genetics in murine norovirus (MNV), demonstrating that a recombinant MNV strain with a single I109V substitution in the protease also showed reduced susceptibility to rupintrivir. In summary, using a combination of different approaches, we have demonstrated that, under the correct conditions, mutations in the norovirus protease that lead to the generation of resistant mutants can rapidly occur.

scholarly journals A Reverse Genetics System for Cypovirus Based on a Bacmid Expressing T7 RNA Polymerase

Viruses ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 314 ◽  
Author(s):  
Gaobo Zhang ◽  
Jian Yang ◽  
Fujun Qin ◽  
Congrui Xu ◽  
Jia Wang ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Reverse Genetics ◽  
T7 Rna Polymerase ◽  
Sf9 Cells ◽  
Genomic Segment ◽  
Egfp Gene ◽  
A Genome ◽  
Typical Morphology ◽  
Occlusion Bodies ◽  
Rescue System

Dendrolimus punctatus cypovirus (DpCPV), belonging to the genus Cypovirus within the family Reoviridae, is considered the most destructive pest of pine forests worldwide. DpCPV has a genome consisting of 10 linear double-stranded RNA segments. To establish a reverse genetics system, we cloned cDNAs encoding the 10 genomic segments of DpCPV into three reverse genetics vectors in which each segment was transcribed under the control of a T7 RNA polymerase promoter and terminator tagged with a hepatitis delta virus ribozyme sequence. We also constructed a vp80-knockout Autographa californica multiple nucleopolyhedrovirus bacmid to express a T7 RNA polymerase codon-optimized for Sf9 cells. Following transfection of Sf9 cells with the three vectors and the bacmid, occlusion bodies (OBs) with the typical morphology of cypovirus polyhedra were observed by optical microscopy. The rescue system was verified by incorporation of a HindIII restriction enzyme site null mutant of the 9th genomic segment. Furthermore, when we co-transfected Sf9 cells with the reverse genetics vectors, the bacmid, and an additional vector bearing an egfp gene flanked with the 5′ and 3′ untranslated regions of the 10th genomic segment, aggregated green fluorescence co-localizing with the OBs was observed. The rescued OBs were able to infect Spodopetra exigua larvae, although their infectivity was significantly lower than that of wild-type DpCPV. This reverse genetics system for DpCPV could be used to explore viral replication and pathogenesis and to facilitate the development of novel bio-insecticides and expression systems for exogenous proteins.

scholarly journals Recovery of Avian Metapneumovirus Subgroup C from cDNA: Cross-Recognition of Avian and Human Metapneumovirus Support Proteins

2006 ◽  
Vol 80 (12) ◽  
pp. 5790-5797 ◽  
Author(s):  
Dhanasekaran Govindarajan ◽  
Ursula J. Buchholz ◽  
Siba K. Samal
Keyword(s):  
Rna Polymerase ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Viral Vector ◽  
The United States ◽  
Human Metapneumovirus ◽  
T7 Rna Polymerase ◽  
Foreign Protein ◽  
Economic Losses ◽  
Avian Metapneumovirus

ABSTRACT Avian metapneumovirus (AMPV) causes an acute respiratory disease in turkeys and is associated with “swollen head syndrome” in chickens, contributing to significant economic losses for the U.S. poultry industry. With a long-term goal of developing a better vaccine for controlling AMPV in the United States, we established a reverse genetics system to produce infectious AMPV of subgroup C entirely from cDNA. A cDNA clone encoding the entire 14,150-nucleotide genome of AMPV subgroup C strain Colorado (AMPV/CO) was generated by assembling five cDNA fragments between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme of a transcription plasmid, pBR 322. Transfection of this plasmid, along with the expression plasmids encoding the N, P, M2-1, and L proteins of AMPV/CO, into cells stably expressing T7 RNA polymerase resulted in the recovery of infectious AMPV/CO. Characterization of the recombinant AMPV/CO showed that its growth properties in tissue culture were similar to those of the parental virus. The potential of AMPV/CO to serve as a viral vector was also assessed by generating another recombinant virus, rAMPV/CO-GFP, that expressed the enhanced green fluorescent protein (GFP) as a foreign protein. Interestingly, GFP-expressing AMPV and GFP-expressing human metapneumovirus (HMPV) could be recovered using the support plasmids of either virus, denoting that the genome promoters are conserved between the two metapneumoviruses and can be cross-recognized by the polymerase complex proteins of either virus. These results indicate a close functional relationship between AMPV/CO and HMPV.

scholarly journals A reverse-genetics system for Influenza A virus using T7 RNA polymerase

2007 ◽  
Vol 88 (4) ◽  
pp. 1281-1287 ◽  
Author(s):  
Emmie de Wit ◽  
Monique I. J. Spronken ◽  
Gaby Vervaet ◽  
Guus F. Rimmelzwaan ◽  
Albert D. M. E. Osterhaus ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Influenza A Virus ◽  
Influenza A ◽  
Reverse Genetics ◽  
T7 Rna Polymerase ◽  
Influenza Virus A ◽  
Sense Orientation ◽  
Polymerase I ◽  
Rna Polymerase Promoter

The currently available reverse-genetics systems for Influenza A virus are all based on transcription of genomic RNA by RNA polymerase I, but the species specificity of this polymerase is a disadvantage. A reverse-genetics vector containing a T7 RNA polymerase promoter, hepatitis delta virus ribozyme sequence and T7 RNA polymerase terminator sequence has been developed. To achieve optimal expression in minigenome assays, it was determined that viral RNA should be inserted in this vector in the negative-sense orientation with two additional G residues downstream of the T7 RNA polymerase promoter. It was also shown that expression of the minigenome was more efficient when a T7 RNA polymerase with a nuclear-localization signal was used. By using this reverse-genetics system, recombinant influenza virus A/PR/8/34 was produced more efficiently than by using a similar polymerase I-based reverse-genetics system. Furthermore, influenza virus A/NL/219/03 could be rescued from 293T, MDCK and QT6 cells. Thus, a reverse-genetics system for the rescue of Influenza A virus has been developed, which will be useful for fundamental research and vaccine seed strain production in a variety of cell lines.

scholarly journals Reverse Genetics System for the Avian Coronavirus Infectious Bronchitis Virus

2001 ◽  
Vol 75 (24) ◽  
pp. 12359-12369 ◽  
Author(s):  
Rosa Casais ◽  
Volker Thiel ◽  
Stuart G. Siddell ◽  
David Cavanagh ◽  
Paul Britton
Keyword(s):  
Molecular Biology ◽  
Rna Polymerase ◽  
Vaccinia Virus ◽  
Infectious Bronchitis Virus ◽  
Reverse Genetics ◽  
Rna Virus ◽  
Full Length ◽  
T7 Rna Polymerase ◽  
Infectious Bronchitis

ABSTRACT Major advances in the study of the molecular biology of RNA viruses have resulted from the ability to generate and manipulate full-length genomic cDNAs of the viral genomes with the subsequent synthesis of infectious RNA for the generation of recombinant viruses. Coronaviruses have the largest RNA virus genomes and, together with genetic instability of some cDNA sequences in Escherichia coli, this has hampered the generation of a reverse-genetics system for this group of viruses. In this report, we describe the assembly of a full-length cDNA from the positive-sense genomic RNA of the avian coronavirus, infectious bronchitis virus (IBV), an important poultry pathogen. The IBV genomic cDNA was assembled immediately downstream of a T7 RNA polymerase promoter by in vitro ligation and cloned directly into the vaccinia virus genome. Infectious IBV RNA was generated in situ after the transfection of restricted recombinant vaccinia virus DNA into primary chick kidney cells previously infected with a recombinant fowlpox virus expressing T7 RNA polymerase. Recombinant IBV, containing two marker mutations, was recovered from the transfected cells. These results describe a reverse-genetics system for studying the molecular biology of IBV and establish a paradigm for generating genetically defined vaccines for IBV.

scholarly journals A plasmid-based reverse genetics system for mammalian orthoreoviruses driven by a plasmid-encoded T7 RNA polymerase

2014 ◽  
Vol 196 ◽  
pp. 36-39 ◽  
Author(s):  
Satoshi Komoto ◽  
Takahiro Kawagishi ◽  
Takeshi Kobayashi ◽  
Mine Ikizler ◽  
Jason Iskarpatyoti ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Reverse Genetics ◽  
T7 Rna Polymerase

scholarly journals African Swine Fever Virus NP868R Capping Enzyme Promotes Reovirus Rescue during Reverse Genetics by Promoting Reovirus Protein Expression, Virion Assembly, and RNA Incorporation into Infectious Virions

2017 ◽  
Vol 91 (11) ◽  
Author(s):  
Heather E. Eaton ◽  
Takeshi Kobayashi ◽  
Terence S. Dermody ◽  
Randal N. Johnston ◽  
Philippe H. Jais ◽  
...  
Keyword(s):  
Protein Expression ◽  
Rna Polymerase ◽  
Reverse Genetics ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Protein Translation ◽  
Fever Virus ◽  
T7 Rna Polymerase ◽  
Capping Enzyme ◽  
Reovirus Replication

ABSTRACT Reoviruses, like many eukaryotic viruses, contain an inverted 7-methylguanosine (m7G) cap linked to the 5′ nucleotide of mRNA. The traditional functions of capping are to promote mRNA stability, protein translation, and concealment from cellular proteins that recognize foreign RNA. To address the role of mRNA capping during reovirus replication, we assessed the benefits of adding the African swine fever virus NP868R capping enzyme during reovirus rescue. C3P3, a fusion protein containing T7 RNA polymerase and NP868R, was found to increase protein expression 5- to 10-fold compared to T7 RNA polymerase alone while enhancing reovirus rescue from the current reverse genetics system by 100-fold. Surprisingly, RNA stability was not increased by C3P3, suggesting a direct effect on protein translation. A time course analysis revealed that C3P3 increased protein synthesis within the first 2 days of a reverse genetics transfection. This analysis also revealed that C3P3 enhanced processing of outer capsid μ1 protein to μ1C, a previously described hallmark of reovirus assembly. Finally, to determine the rate of infectious-RNA incorporation into new virions, we developed a new recombinant reovirus S1 gene that expressed the fluorescent protein UnaG. Following transfection of cells with UnaG and infection with wild-type virus, passage of UnaG through progeny was significantly enhanced by C3P3. These data suggest that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation. IMPORTANCE Our findings expand our understanding of how viruses utilize capping, suggesting that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation, in addition to enhancing protein translation. Beyond providing mechanistic insight into reovirus replication, our findings also show that reovirus reverse genetics rescue is enhanced 100-fold by the NP868R capping enzyme. Since reovirus shows promise as a cancer therapy, efficient reovirus reverse genetics rescue will accelerate production of recombinant reoviruses as candidates to enhance therapeutic potency. NP868R-assisted reovirus rescue will also expedite production of recombinant reovirus for mechanistic insights into reovirus protein function and structure.

scholarly journals Regeneration of Gardenia gummifera Linn.f by using cyanobacteria- A novel approach to tissue culture

2019 ◽  
Vol 8 (1) ◽  
pp. 3489
Author(s):  
Firdous Mir ◽  
Zakir Hussain Khanday ◽  
Sumer Singh
Keyword(s):  
Tissue Culture ◽  
Genetic Variation ◽  
Medicinal Plants ◽  
Normal Tissue ◽  
Low Cost ◽  
Cost Effective ◽  
Culture Technique ◽  
Tissue Culture Technique ◽  
Novel Approach ◽  
First Time

Propagation of the medicinal plants by usage of different media and PGR’s is laborious, cost-effective and is the possibility of genetic variation. In the present investigation, a novel protocol was first time developed for propagation of Gardenia gummifera Linn.f. This protocol is useful in all aspects viz low cost, time and free from genetic variation. This technology is efficient as compared to normal tissue culture technique which is used for conservation from last of two decades.

scholarly journals Efficient cDNA-Based Rescue of La Crosse Bunyaviruses Expressing or Lacking the Nonstructural Protein NSs

2005 ◽  
Vol 79 (16) ◽  
pp. 10420-10428 ◽  
Author(s):  
Gjon Blakqori ◽  
Friedemann Weber
Keyword(s):  
Rna Polymerase ◽  
Reverse Genetics ◽  
Nonstructural Protein ◽  
Viral Proteins ◽  
Vero Cells ◽  
T7 Rna Polymerase ◽  
Wild Type Virus ◽  
La Crosse Virus ◽  
Additional Support ◽  
Bunyamwera Virus

ABSTRACT La Crosse virus (LACV) belongs to the Bunyaviridae family and causes severe encephalitis in children. It has a negative-sense RNA genome which consists of the three segments L, M, and S. We successfully rescued LACV by transfection of just three plasmids, using a system which was previously established for Bunyamwera virus (Lowen et al., Virology 330:493-500, 2004). These cDNA plasmids represent the three viral RNA segments in the antigenomic orientation, transcribed intracellularly by the T7 RNA polymerase and with the 3′ ends trimmed by the hepatitis delta virus ribozyme. As has been shown for Bunyamwera virus, the antigenomic plasmids could serve both as donors for the antigenomic RNA and as support plasmids to provide small amounts of viral proteins for RNA encapsidation and particle formation. In contrast to other rescue systems, however, transfection of additional support plasmids completely abrogated the rescue, indicating that LACV is highly sensitive to overexpression of viral proteins. The BSR-T7/5 cell line, which constitutively expresses T7 RNA polymerase, allowed efficient rescue of LACV, generating approximately 108 infectious viruses per milliliter. The utility of this system was demonstrated by the generation of a wild-type virus containing a genetic marker (rLACV) and of a mutant with a deleted NSs gene on the S segment (rLACVdelNSs). The NSs-expressing rLACV formed clear plaques, displayed an efficient host cell shutoff, and was strongly proapoptotic. The rLACVdelNSs mutant, by contrast, exhibited a turbid-plaque phenotype and a less-pronounced shutoff and induced little apoptosis. Nevertheless, both viruses grew in Vero cells to similar titers. Our reverse genetics system now enables us to manipulate the genome of LACV in order to characterize its virulence factors and to develop potential vaccine candidates.

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