scholarly journals Pathogen-derived resistance to dengue type 2 virus in mosquito cells by expression of the premembrane coding region of the viral genome.

1996 ◽  
Vol 70 (4) ◽  
pp. 2132-2137 ◽  
Author(s):  
P J Gaines ◽  
K E Olson ◽  
S Higgs ◽  
A M Powers ◽  
B J Beaty ◽  
...  
Keyword(s):  
Viral Genome ◽  
Coding Region ◽  
Mosquito Cells ◽  
Pathogen Derived Resistance ◽  
Dengue Type 2 Virus

scholarly journals Substitution or deletion mutations between nt 54 and 70 in the 5′ non-coding region of dengue type 2 virus produce variable effects on virus viability

2007 ◽  
Vol 88 (6) ◽  
pp. 1748-1752 ◽  
Author(s):  
Wipawan Sirigulpanit ◽  
Richard M. Kinney ◽  
Vijittra Leardkamolkarn
Keyword(s):  
Infectious Clone ◽  
Single Point ◽  
Serial Passage ◽  
Coding Region ◽  
Deletion Mutations ◽  
Nucleotide Mutation ◽  
Virus Produce ◽  
Dengue Type 2 Virus ◽  
Stem Structure

A C57U nucleotide mutation in a predicted RNA stem structure (nt 11–16/56–61) of the 5′ non-coding region (5′NCR) of dengue 2 (DEN-2) 16681 virus is partially attenuating, but unstable during serial passage of certain candidate DEN-2 PDK-53-based vaccine viruses containing this mutation. Here, 11 different mutations (one or more point substitution and/or deletion) between nt 54 and 70 in the 5′NCR of the pD2/IC-30P-A (16681) infectious clone are described. Four mutants were infectious. Three mutants with single point substitutions replicated well in cell culture and exhibited variable neurovirulence in mice. Constructs containing multiple substitutions or any deletions failed to produce infectious viruses. Unexpectedly, a double C57U+G58C mutant replicated as efficiently as D2/IC-30P-A virus, and was more neurovirulent for newborn ICR mice. Thus, despite its predicted additional disruption of the RNA stem structure, the engineered contiguous secondary G58C mutation caused reversion of the partially attenuated phenotype caused by the 5′NCR-C57U mutation.

Generation and preclinical immunogenicity study of dengue type 2 virus-like particles derived from stably transfected mosquito cells

Vaccine ◽  
2015 ◽  
Vol 33 (42) ◽  
pp. 5613-5622 ◽  
Author(s):  
Amporn Suphatrakul ◽  
Thippawan Yasanga ◽  
Poonsook Keelapang ◽  
Rungtawan Sriburi ◽  
Thaneeya Roytrakul ◽  
...  
Keyword(s):  
Virus Like Particles ◽  
Mosquito Cells ◽  
Dengue Type 2 Virus ◽  
Immunogenicity Study

scholarly journals RNA Secondary Structure in the Coding Region of Dengue Virus Type 2 Directs Translation Start Codon Selection and Is Required for Viral Replication

2006 ◽  
Vol 80 (5) ◽  
pp. 2170-2182 ◽  
Author(s):  
Karen Clyde ◽  
Eva Harris
Keyword(s):  
Dengue Virus ◽  
Viral Replication ◽  
Virus Type ◽  
Start Codon ◽  
Coding Region ◽  
Mosquito Cells ◽  
Translation Start ◽  
Codon Selection ◽  
Dengue Virus Type 2

ABSTRACT Dengue virus is a positive-strand RNA virus and a member of the genus Flavivirus, which includes West Nile, yellow fever, and tick-borne encephalitis viruses. Flavivirus genomes are translated as a single polyprotein that is subsequently cleaved into 10 proteins, the first of which is the viral capsid (C) protein. Dengue virus type 2 (DENV2) and other mosquito-borne flaviviruses initiate translation of C from a start codon in a suboptimal context and have multiple in-frame AUGs downstream. Here, we show that an RNA hairpin structure in the capsid coding region (cHP) directs translation start site selection in human and mosquito cells. The ability of the cHP to direct initiation from the first start codon is proportional to its thermodynamic stability, is position dependent, and is sequence independent, consistent with a mechanism in which the scanning initiation complex stalls momentarily over the first AUG as it begins to unwind the cHP. The cHP of tick-borne flaviviruses is not maintained in a position to influence start codon selection, which suggests that this coding region cis element may serve another function in the flavivirus life cycle. Here, we demonstrate that the DENV2 cHP and both the first and second AUGs of C are necessary for efficient viral replication in human and mosquito cells. While numerous regulatory elements have been identified in the untranslated regions of RNA viral genomes, we show that the cHP is a coding-region RNA element that directs start codon selection and is required for viral replication.

scholarly journals Systemic Polyomavirus Genome Increase and Dissemination of Capsid-Defective Genomes in Mammary Gland Tumor-Bearing Mice

2000 ◽  
Vol 74 (15) ◽  
pp. 6975-6983 ◽  
Author(s):  
Julie J. Wirth ◽  
Li Chen ◽  
Michele M. Fluck
Keyword(s):  
Mammary Gland ◽  
Viral Genome ◽  
Wild Type Strain ◽  
Neonatal Infection ◽  
Wild Type ◽  
Coding Region ◽  
Live Virus ◽  
Viral Genomes ◽  
Mammary Gland Tumors ◽  
Low Levels

ABSTRACT BALB/c mice that developed tumors 7 to 8 months following neonatal infection by polyomavirus (PYV) wild-type strain A2 were characterized with respect to the abundance and integrity of the viral genome in the tumors and in 12 nontumorous organs. These patterns were compared to those found in tumor-free mice infected in parallel. Six mice were analyzed in detail including four sibling females with mammary gland tumors. In four of five mammary gland tumors, the viral genome had undergone a unique deletion and/or rearrangement. Three tumor-resident genomes with an apparently intact large T coding region were present in abundant levels in an unintegrated state. Two of these had undergone deletions and rearrangements involving the capsid genes and therefore lacked the capacity to produce live virus. In the comparative organ survey, the tumors harboring replication-competent genomes contained by far the highest levels of genomes of any tissue. However, the levels of PYV genomes in other organs were elevated by up to 1 to 2 orders of magnitude compared to those detected in the same organs of tumor-free mice. The genomes found in the nontumorous organs had the same rearrangements as the genomes residing in the tumors. The original wild-type genome was detected at low levels in a few organs, particularly in the kidneys. The data indicate that a systemic increase in the level of viral genomes occurred in conjunction with the induction of tumors by PYV. The results suggest two novel hypotheses: (i) that genomes may spread from the tumors to the usual PYV target tissues and (ii) that this dissemination may take place in the absence of capsids, providing an important path for a virus to escape from the immune response. This situation may offer a useful model for the spread of HPV accompanying HPV-induced oncogenesis.

scholarly journals Influenza virus-susceptible mice carry Mx genes with a large deletion or a nonsense mutation.

1988 ◽  
Vol 8 (10) ◽  
pp. 4518-4523 ◽  
Author(s):  
P Staeheli ◽  
R Grob ◽  
E Meier ◽  
J G Sutcliffe ◽  
O Haller
Keyword(s):  
Influenza Virus ◽  
Influenza Virus Infection ◽  
Cdna Clones ◽  
Mrna Levels ◽  
Coding Region ◽  
Hindiii Site ◽  
Rflp Type ◽  
Mx Protein ◽  
Type 3

The interferon-regulated mouse Mx gene encodes the 72-kilodalton nuclear Mx protein that selectively inhibits influenza virus replication. Mice carrying Mx+ alleles synthesize Mx protein and resist influenza virus infection, whereas mice homozygous for Mx- alleles fail to synthesize Mx protein and, as a consequence, are influenza virus susceptible. Southern blot analysis allowed us to define the following three distinct Mx restriction fragment length polymorphism (RFLP) types among classical inbred strains: RFLP type 1 in the Mx+ strains A2G and SL/NiA, RFLP type 2 in BALB/c and 33 other Mx- strains, and RFLP type 3 in CBA/J and 2 other Mx- strains. cDNA clones of Mx mRNAs from BALB/c and CBA/J cells were isolated, and their sequences were compared with that of the wild-type Mx mRNA of strain A2G. Mx mRNA of BALB/c mice has 424 nucleotides absent from the coding region, resulting in a frame shift and premature termination of Mx protein. The missing sequences correspond exactly to Mx exons 9 through 11. These three exons, together with some flanking intron sequences, are deleted from the genomes of all Mx RFLP type 2 strains. The Mx- phenotype of the Mx RFLP type 3 strain CBA/J is due to a point mutation that converts the lysine codon in position 389 to a termination codon. Mx RFLP type 3 strains have an extra HindIII site which maps to an intron and thus probably does not affect the coding capacity of Mx mRNA. We further show that the Mx mRNA levels in interferon-treated BALB/c and CBA/J cells are about 15-fold lower than in similarly treated Mx+ cells. This is probably due to decreased metabolic stabilities of the mutant mRNAs.

scholarly journals Aedes albopictus (Skuse, 1894) infected with the American-Asian genotype of dengue type 2 virus in Medellín suggests its possible role as vector of dengue fever in Colombia

Biomédica ◽  
2017 ◽  
Vol 37 ◽  
pp. 135 ◽  
Author(s):  
Andrés Gómez-Palacio ◽  
Juan Suaza-Vasco ◽  
Sandra Castaño ◽  
Omar Triana ◽  
Sandra Uribe
Keyword(s):  
Aedes Aegypti ◽  
Dengue Fever ◽  
Aedes Albopictus ◽  
Distribución Geográfica ◽  
Dengue Type 2 Virus

Introducción. Aedes aegypti y Ae. albopictus son reconocidos vectores de arbovirus como los del dengue, la fiebre amarilla, el chikungunya y el Zika, en regiones tropicales y subtropicales del mundo. En Colombia, la distribución geográfica de Ae. albopictus ha sufrido un incremento y hoy incluye ciudades como Cali y Medellín. Hasta ahora, sin embargo, no se ha recabado información concluyente sobre su infección viral y su capacidad de transmisión a los humanos.Objetivo. Determinar la infección natural por dengue en ejemplares de Ae. albopictus recolectados en un área urbana de Medellín.Materiales y métodos. Se recolectaron individuos de Ae. albopictus en el campus de la Universidad Nacional de Colombia, sede Medellín. Se confirmó su clasificación taxonómica mediante el análisis del gen citocromo oxidasa I (COI), y se extrajo el ARN total para la identificación del virus del dengue y de los respectivos serotipos. La presencia del genotipo DENV se infirió mediante el análisis del gen NS3.Resultados. El análisis del COI corroboró el estatus taxonómico de Ae. albopictus. Uno de los mosquitos procesados fue positivo para DENV-2 y el análisis del NS3 mostró una gran similitud con el genotipo asiático-americano.Conclusión. Se reporta la infección con DENV-2 en Ae. albopictus en Medellín, Colombia. La presencia del genotipo asiático-americano en una zona urbana sugiere su posible circulación entre humanos y en Ae. albopictus, lo cual alerta sobre su eventual papel en la transmisión del DENV-2, y sobre la necesidad de incluir esta especie en la vigilancia entomológica en Colombia.

scholarly journals ANALYSIS OF THE ASSOCIATION OF THE METHYLATION LEVELS OF MIR10B AND MIR21 GENES IN BLOOD LEUKOCYTES WITH ADVANCED CAROTID ATHEROSCLEROSIS

2018 ◽  
Vol 33 (2) ◽  
pp. 77-82
Author(s):  
Iu. A. Koroleva ◽  
A. A. Zarubin ◽  
A. V. Markov ◽  
A. N. Kazancev ◽  
O. L. Barbarash ◽  
...  
Keyword(s):  
Risk Factors ◽  
Dna Methylation ◽  
Methylation Level ◽  
Carotid Atherosclerosis ◽  
Gene Promoter ◽  
Control Group ◽  
Coding Region ◽  
Blood Leukocytes ◽  
Atherosclerosis Risk Factors

Complications of atherosclerosis remain the leading cause of morbidity and mortality worldwide. MiRNAs are short regulatory molecules that are involved in all processes of pathogenesis. Expression of miRNAs is regulated by DNA methylation. Methylation and/or expression of MIR10B and MIR21 genes are known to vary in atherosclerotic tissues of the arteries, but there is no data about the changes in the methylation levels of these genes in blood leukocytes and their association with atherosclerosis risk factors.Objective.To evaluate the association of methylation levels of MIR10B and MIR21 genes in the blood leukocytes with risk factors and pathogenetically significant traits of carotid atherosclerosis.Material and Methods. DNA for the study was extracted from the samples of blood leukocytes of 122 patients with advanced carotid atherosclerosis as well as from blood leukocytes of 135 individuals in the control group. The DNA methylation level was analyzed by bisulfite pyrosequencing.Results.The methylation level of the MIR10B and MIR21 genes in leukocytes of patients with atherosclerosis is higher than in the leukocytes of the control group. In leukocytes of patients with carotid atherosclerosis the methylation level of the MIR21 gene promoter was correlated with type 2 diabetes and serum cholesterol level, and the methylation level of the coding region of the MIR10B gene was correlated with smoking.Conclusions.The level of DNA methylation in the regions of MIR10B and MIR21 genes in blood leukocytes is associated with the risk of advanced atherosclerosis of the carotid arteries. 

scholarly journals Mosquito metabolomics reveal that dengue virus replication requires phospholipid reconfiguration via the remodeling cycle

2020 ◽  
Vol 117 (44) ◽  
pp. 27627-27636
Author(s):  
Thomas Vial ◽  
Wei-Lian Tan ◽  
Eric Deharo ◽  
Dorothée Missé ◽  
Guillaume Marti ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Viral Genome ◽  
De Novo ◽  
Genome Replication ◽  
Mosquito Vector ◽  
Phospholipid Biosynthesis ◽  
Replication Complex ◽  
Mosquito Cells ◽  
Viral Genome Replication ◽  
Blood Meals

Dengue virus (DENV) subdues cell membranes for its cellular cycle by reconfiguring phospholipids in humans and mosquitoes. Here, we determined how and why DENV reconfigures phospholipids in the mosquito vector. By inhibiting and activating the de novo phospholipid biosynthesis, we demonstrated the antiviral impact of de novo–produced phospholipids. In line with the virus hijacking lipids for its benefit, metabolomics analyses indicated that DENV actively inhibited the de novo phospholipid pathway and instead triggered phospholipid remodeling. We demonstrated the early induction of remodeling during infection by using isotope tracing in mosquito cells. We then confirmed in mosquitoes the antiviral impact of de novo phospholipids by supplementing infectious blood meals with a de novo phospholipid precursor. Eventually, we determined that phospholipid reconfiguration was required for viral genome replication but not for the other steps of the virus cellular cycle. Overall, we now propose that DENV reconfigures phospholipids through the remodeling cycle to modify the endomembrane and facilitate formation of the replication complex. Furthermore, our study identified de novo phospholipid precursor as a blood determinant of DENV human-to-mosquito transmission.

scholarly journals Conformational Changes and Nuclear Entry of Porcine Circovirus without Disassembly

2019 ◽  
Vol 93 (20) ◽  
Author(s):  
Huijuan Wang ◽  
Kailun Zhang ◽  
Cui Lin ◽  
Jianwei Zhou ◽  
Yulan Jin ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Viral Genome ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Porcine Circovirus ◽  
Nuclear Targeting ◽  
Early Endosomes ◽  
Pcv2 Genome ◽  
Infectious Cycle

ABSTRACT A relatively stable and flexible capsid is critical to the viral life cycle. However, the capsid dynamics and cytosol trafficking of porcine circovirus type 2 (PCV2) during its infectious cycle are poorly understood. Here, we report the structural stability and conformation flexibility of PCV2 virions by genome labeling and the use of three monoclonal antibodies (MAbs) against the native capsid of PCV2. Genome labeling showed that the infectivity of the PCV2 virion was not affected by conjugation with deoxy-5-ethynylcytidine (EdC). Heat stability experiments indicated that PCV2 capsids started to disassemble at 65°C, causing binding incompetence for all antibodies, and the viral genome was released without capsid disassembly upon heating at 60°C. Antibody binding experiments with PCV2 showed that residues 186 to 192 were concealed in the early endosomes of epithelial PK-15 and monocytic 3D4/31 cells with or without chloroquine treatment and then exposed in PK-15 cytosol and the 3D4/31 nucleus. Viral propagation and localization experiments showed that PCV2 replication and cytosol trafficking were not significantly affected by microtubule depolymerization in monocytic 3D4/31 cells treated with nocodazole. These findings demonstrated that nuclear targeting of viral capsids involved conformational changes, the PCV2 genome was released from the assembled capsid, and the transit of PCV2 particles was independent of microtubules in 3D4/31 cells. IMPORTANCE Circovirus is the smallest virus known to replicate autonomously. Knowledge of viral genome release may provide understanding of viral replication and a method to artificially inactivate viral particles. Currently, little is known about the release model of porcine circovirus type 2 (PCV2). Here, we report the release of the PCV2 genome from assembled capsid and the intracellular trafficking of infectious PCV2 by alterations in the capsid conformation. Knowledge of PCV2 capsid stability and dynamics is essential to understanding its infectious cycle and lays the foundation for discovering powerful targets for therapeutic and prophylactic intervention.

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