Development of an entirely plasmid-based reverse genetics system for 12-segmented double-stranded RNA viruses

2021 ◽  
Vol 118 (42) ◽  
pp. e2105334118
Author(s):  
Ryotaro Nouda ◽  
Shohei Minami ◽  
Yuta Kanai ◽  
Takahiro Kawagishi ◽  
Jeffery A. Nurdin ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Therapeutic Interventions ◽  
Viral Gene ◽  
Kidney Cells ◽  
Virus Species ◽  
Double Stranded Rna ◽  
A Genome ◽  
The Family ◽  
Nonenveloped Virus ◽  
Baby Hamster Kidney Cells

The family Reoviridae is a nonenveloped virus group with a double-stranded (ds) RNA genome comprising 9 to 12 segments. In the family Reoviridae, the genera Cardoreovirus, Phytoreovirus, Seadornavirus, Mycoreovirus, and Coltivirus contain virus species having 12-segmented dsRNA genomes. Reverse genetics systems used to generate recombinant infectious viruses are powerful tools for investigating viral gene function and for developing vaccines and therapeutic interventions. Generally, this methodology has been utilized for Reoviridae viruses such as Orthoreovirus, Orbivirus, Cypovirus, and Rotavirus, which have genomes with 10 or 11 segments, respectively. However, no reverse genetics system has been developed for Reoviridae viruses with a genome harboring 12 segments. Herein, we describe development of an entire plasmid-based reverse genetics system for Tarumizu tick virus (TarTV) (genus Coltivirus, family Reoviridae), which has a genome of 12 segments. Recombinant TarTVs were generated by transfection of 12 cloned complementary DNAs encoding the TarTV genome into baby hamster kidney cells expressing T7 RNA polymerase. Using this technology, we generated VP12 mutant viruses and demonstrated that VP12 is an N-glycosylated protein. We also generated a reporter virus expressing the HiBiT-tagged VP8 protein. This reverse genetics system will increase our understanding of not only the biology of the genus Coltivirus but also the replication machinery of the family Reoviridae.

scholarly journals Double-stranded RNA viruses infecting Epichloe festucae

2007 ◽  
Vol 13 ◽  
pp. 207-209
Author(s):  
M. Romo ◽  
A. Leuchtmann ◽  
B. García ◽  
I. Zabalgogeazcoa
Keyword(s):  
Rna Viruses ◽  
Virus Transmission ◽  
Fungal Endophyte ◽  
Double Stranded Rna ◽  
Dsrna Molecule ◽  
Epichloë Festucae ◽  
A Genome ◽  
The Family ◽  
Two Populations ◽  
Ascospore Formation

Some isolates of Epichloë festucae are asymptomatically infected by Epichloë festucae virus 1 (EfV1), a member of the family Totiviridae. This virus has a genome composed by a 5109 bp molecule of double stranded RNA (dsRNA). In addition, a 3 kbp dsRNA molecule which could be the genome of another virus (EfV2) was frequently found in isolates of the fungal endophyte. In a survey of two populations of E. festucae it was found that 73% of the isolates were infected by one or both viruses. Although both viruses are very efficiently transmitted to conidia, a barrier to virus transmission occurs during ascospore formation. Keywords: Epichloë festucae, virus, dsRNA, Totivirus

Turbomolecular Pumped Electron Microscopy of Rubella Virus (In BHK Celis)

1968 ◽  
Vol 26 ◽  
pp. 204-205
Author(s):  
A. B. Taylor ◽  
G. C. Cole ◽  
M. A. Holcomb ◽  
C. A. Baechler
Keyword(s):  
Electron Microscopy ◽  
Rubella Virus ◽  
Phosphate Buffer ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Basal Medium ◽  
Kidney Cells ◽  
Baby Hamster Kidney ◽  
Input Multiplicity ◽  
Baby Hamster Kidney Cells

An aliquot from a continuous fermenter culture of baby hamster kidney cells (BHK-21 Clone PD-4) (Wistar) maintained in Ca free Eagle's Basal Medium containing 2% Kaolin adsorbed fetal calf serum was planted in spinner flasks at 300,000 cells per ml, total volume 600 ml. After equilibration for one day at 35°C to insure that cells were in log phase, the culture was infected with the M-33-AGMK25 BHK-219 strain of rubella at an input multiplicity of about 6 TCID50 per cell. The virus was identified with specific rubella antiserum.Preliminary experiments had shown that such cultures would reach a peak or plateau HA titer of approximately 1:64, 24 hrs after inoculation and would continue to yield virus for 6 to 12 days. One hundred ml aliquot harvests were withdrawn daily and the culture was returned to volume with growth medium and incubation continued. The harvested cells were spun down rapidly at 2500 rpm per 15 mins., fixed in 3.7% gluteraldehyde in Ca free phosphate buffer saline, and post fixed in osmium tetraoxide. After dehydration, the cells were embedded in Epon 812 and cured approximately 20 hrs at 60°C.

scholarly journals The [KIL-d] Element Specifically Regulates Viral Gene Expression in Yeast

Genetics ◽  
2000 ◽  
Vol 155 (2) ◽  
pp. 601-609 ◽  
Author(s):  
Zsolt Tallóczy ◽  
Rebecca Mazar ◽  
Denise E Georgopoulos ◽  
Fausto Ramos ◽  
Michael J Leibowitz
Keyword(s):  
Gene Expression ◽  
Phenotypic Expression ◽  
Viral Gene Expression ◽  
Virus Genome ◽  
Viral Rna ◽  
High Rate ◽  
Viral Gene ◽  
Double Stranded Rna ◽  
Yeast Prions ◽  
Killer Virus

Abstract The cytoplasmically inherited [KIL-d] element epigenetically regulates killer virus gene expression in Saccharomyces cerevisiae. [KIL-d] results in variegated defects in expression of the M double-stranded RNA viral segment in haploid cells that are “healed” in diploids. We report that the [KIL-d] element is spontaneously lost with a frequency of 10−4–10−5 and reappears with variegated phenotypic expression with a frequency of ≥10−3. This high rate of loss and higher rate of reappearance is unlike any known nucleic acid replicon but resembles the behavior of yeast prions. However, [KIL-d] is distinct from the known yeast prions in its relative guanidinium hydrochloride incurability and independence of Hsp104 protein for its maintenance. Despite its transmissibility by successive cytoplasmic transfers, multiple cytoplasmic nucleic acids have been proven not to carry the [KIL-d] trait. [KIL-d] epigenetically regulates the expression of the M double-stranded RNA satellite virus genome, but fails to alter the expression of M cDNA. This specificity remained even after a cycle of mating and meiosis. Due to its unique genetic properties and viral RNA specificity, [KIL-d] represents a new type of genetic element that interacts with a viral RNA genome.

scholarly journals Extensive Phylogenetic Analysis of Piscine Orthoreovirus Genomic Sequences Shows the Robustness of Subgenotype Classification

Pathogens ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 41
Author(s):  
Marcos Godoy ◽  
Daniel A. Medina ◽  
Rudy Suarez ◽  
Sandro Valenzuela ◽  
Jaime Romero ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Genetic Variation ◽  
Sequence Analysis ◽  
Molecular Phylogeny ◽  
Phylogenetic Trees ◽  
Genomic Segment ◽  
Double Stranded Rna ◽  
The Family ◽  
Original Classification

Piscine orthoreovirus (PRV) belongs to the family Reoviridae and has been described mainly in association with salmonid infections. The genome of PRV consists of about 23,600 bp, with 10 segments of double-stranded RNA, classified as small (S1 to S4), medium (M1, M2 and M3) and large (L1, L2 and L3); these range approximately from 1000 bp (segment S4) to 4000 bp (segment L1). How the genetic variation among PRV strains affects the virulence for salmonids is still poorly understood. The aim of this study was to describe the molecular phylogeny of PRV based on an extensive sequence analysis of the S1 and M2 segments of PRV available in the GenBank database to date (May 2020). The analysis was extended to include new PRV sequences for S1 and M2 segments. In addition, subgenotype classifications were assigned to previously published unclassified sequences. It was concluded that the phylogenetic trees are consistent with the original classification using the PRV genomic segment S1, which differentiates PRV into two major genotypes, I and II, and each of these into two subgenotypes, designated as Ia and Ib, and IIa and IIb, respectively. Moreover, some clusters of country- and host-specific PRV subgenotypes were observed in the subset of sequences used. This work strengthens the subgenotype classification of PRV based on the S1 segment and can be used to enhance research on the virulence of PRV.

scholarly journals Knockdown of LIM15/DMC1 in the mushroom Coprinus cinereus by double-stranded RNA-mediated gene silencing

Microbiology ◽  
2005 ◽  
Vol 151 (11) ◽  
pp. 3669-3678 ◽  
Author(s):  
Satoshi H. Namekawa ◽  
Kazuki Iwabata ◽  
Hiroko Sugawara ◽  
Fumika N. Hamada ◽  
Akiyo Koshiyama ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Sexual Development ◽  
Reverse Genetics ◽  
Model Organism ◽  
Coprinus Cinereus ◽  
Gene Repression ◽  
Mushroom Species ◽  
Double Stranded Rna ◽  
Chromosome Synapsis ◽  
Expression Construct

The basidiomycete Coprinus cinereus has many advantages as a model organism for studying sexual development and meiosis, but it has been difficult to investigate using reverse-genetics methods, such as gene disruption by homologous recombination. Here, gene repression by dsRNA-mediated gene silencing was tried as an alternative method for reverse-genetics studies. It was shown that transformation of the LIM15/DMC1 dsRNA expression construct (LIM15dsRNA) resulted in genomic insertion of LIM15dsRNA and paucity of the LIM15/DMC1 transcript. First, LIM15dsRNA was transformed into the homothallic strain AmutBmut to generate a homozygote in which both nuclei had a copy of LIM15dsRNA. The LIM15/DMC1-repressed strain showed abnormal homologous chromosome synapsis during meiosis. Basidiospore production was reduced to 16 % by the induction of dsRNA. However, approximately 60 % of basidiospores were viable. Next, a heterozygote was generated in which one nucleus had a copy of LIM15dsRNA. The phenotype was similar to that of the homozygote. These results are not only the first demonstration of dsRNA-mediated gene silencing in a member of the homobasidiomycete fungi, to which 90 % of mushroom species belong, but also the first successful use of a reverse-genetics approach in C. cinereus research.

scholarly journals Allicin from garlic strongly inhibits cysteine proteinases and cytopathic effects of Entamoeba histolytica.

1997 ◽  
Vol 41 (10) ◽  
pp. 2286-2288 ◽  
Author(s):  
S Ankri ◽  
T Miron ◽  
A Rabinkov ◽  
M Wilchek ◽  
D Mirelman
Keyword(s):  
Alcohol Dehydrogenase ◽  
Entamoeba Histolytica ◽  
Kidney Cells ◽  
Cysteine Proteinases ◽  
Baby Hamster Kidney ◽  
Important Contributor ◽  
Cytopathic Effects ◽  
Baby Hamster Kidney Cells

The ability of Entamoeba histolytica trophozoites to destroy monolayers of baby hamster kidney cells is inhibited by allicin, one of the active principles of garlic. Cysteine proteinases, an important contributor to amebic virulence, as well as alcohol dehydrogenase, are strongly inhibited by allicin.

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