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.

scholarly journals Genetic Determinants Responsible for Acquisition of Dengue Type 2 Virus Mouse Neurovirulence

1998 ◽  
Vol 72 (2) ◽  
pp. 1647-1651 ◽  
Author(s):  
Michael Bray ◽  
Ruhe Men ◽  
Issei Tokimatsu ◽  
Ching-Juh Lai
Keyword(s):  
Amino Acid ◽  
Serial Passage ◽  
Minor Effect ◽  
Genetic Determinants ◽  
Dengue Viruses ◽  
Ns1 Gene ◽  
Significant Attenuation ◽  
A Minor ◽  
Dengue Type 2 Virus

ABSTRACT Studies conducted some 50 years ago showed that serial intracerebral passage of dengue viruses in mice selected for neurovirulent mutants that also exhibited significant attenuation for humans. We investigated the genetic basis of mouse neurovirulence of dengue virus because it might be directly or indirectly associated with attenuation for humans. Analysis of the sequence in the C-PreM-E-NS1 region of the parental dengue type 2 virus (DEN2) New Guinea C (NGC) strain and its mouse-adapted, neurovirulent mutant revealed that 10 nucleotide changes occurred during serial passage in mice. Seven of these changes resulted in amino acid substitutions, i.e., Leu55-Phe and Arg57-Lys in PreM, Glu71-Asp, Glu126-Lys, Phe402-Ile, and Thr454-Ile in E, and Arg105-Gln in NS1. The sequence of C was fully conserved between the parental and mutant DEN2. We constructed intertypic chimeric dengue viruses that contained the PreM-E genes or only the NS1 gene of neurovirulent DEN2 NGC substituting for the corresponding genes of DEN4. The DEN2 (PreM-E)/DEN4 chimera was neurovirulent for mice, whereas DEN2 (NS1)/DEN4 was not. The mutations present in the neurovirulent DEN2 PreM-E genes were then substituted singly or in combination into the sequence of the nonneurovirulent, parental DEN2. Intracerebral titration of the various mutant chimeras so produced identified two amino acid changes, namely, Glu71-Asp and Glu126-Lys, in DEN2 E as being responsible for mouse neurovirulence. The conservative amino acid change of Glu71-Asp probably had a minor effect, if any. The Glu126-Lys substitution in DEN2 E, representing a change from a negatively charged amino acid to a positively charged amino acid, most likely plays an important role in conferring mouse neurovirulence.

scholarly journals Genetic Inactivation of Poliovirus Infectivity by Increasing the Frequencies of CpG and UpA Dinucleotides within and across Synonymous Capsid Region Codons

2009 ◽  
Vol 83 (19) ◽  
pp. 9957-9969 ◽  
Author(s):  
Cara C. Burns ◽  
Ray Campagnoli ◽  
Jing Shaw ◽  
Annelet Vincent ◽  
Jaume Jorba ◽  
...  
Keyword(s):  
Infectious Clone ◽  
Prototype Strain ◽  
Single Step ◽  
Temperature Sensitive ◽  
Coding Region ◽  
Capsid Region ◽  
Replicative Fitness ◽  
Synonymous Codons ◽  
Effective Number Of Codons

ABSTRACT Replicative fitness of poliovirus can be modulated systematically by replacement of preferred capsid region codons with synonymous unpreferred codons. To determine the key genetic contributors to fitness reduction, we introduced different sets of synonymous codons into the capsid coding region of an infectious clone derived from the type 2 prototype strain MEF-1. Replicative fitness in HeLa cells, measured by plaque areas and virus yields in single-step growth experiments, decreased sharply with increased frequencies of the dinucleotides CpG (suppressed in higher eukaryotes and most RNA viruses) and UpA (suppressed nearly universally). Replacement of MEF-1 capsid codons with the corresponding codons from another type 2 prototype strain (Lansing), a randomization of MEF-1 synonymous codons, increased the %G+C without increasing CpG, and reductions in the effective number of codons used had much smaller individual effects on fitness. Poliovirus fitness was reduced to the threshold of viability when CpG and UpA dinucleotides were saturated within and across synonymous codons of a capsid region interval representing only ∼9% of the total genome. Codon replacements were associated with moderate decreases in total virion production but large decreases in the specific infectivities of intact poliovirions and viral RNAs. Replication of codon replacement viruses, but not MEF-1, was temperature sensitive at 39.5°C. Synthesis and processing of viral intracellular proteins were largely unaltered in most codon replacement constructs. Replacement of natural codons with synonymous codons with increased frequencies of CpG and UpA dinucleotides may offer a general approach to the development of attenuated vaccines with well-defined antigenicities and very high genetic stabilities.

scholarly journals OsPRR37 Alternatively Promotes Heading Date Through Suppressing the Expression of Ghd7 in the Japonica Variety Zhonghua 11 under Natural Long-Day Conditions

Rice ◽  
2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Yong Hu ◽  
Xin Zhou ◽  
Bo Zhang ◽  
Shuangle Li ◽  
Xiaowei Fan ◽  
...  
Keyword(s):  
Functional Divergence ◽  
Heading Date ◽  
Transcriptional Analysis ◽  
Dependent Manner ◽  
Coding Region ◽  
Japonica Variety ◽  
Long Day ◽  
Nucleotide Mutation ◽  
Alternative Function ◽  
Pr Domain

AbstractHeading date is an important agronomic trait of rice (Oryza sativa L.) and is regulated by numerous genes, some of which exhibit functional divergence in a genetic background-dependent manner. Here, we identified a late heading date 7 (lhd7) mutant that flowered later than wild-type Zhonghua 11 (ZH11) under natural long-day (NLD) conditions. Map-based cloning facilitated by the MutMap strategy revealed that LHD7 was on the same locus as OsPRR37 but exhibited a novel function as a promoter of heading date. A single-nucleotide mutation of G-to-A in the coding region caused a substitution of aspartic acid for glycine at site 159 within the pseudo-receiver (PR) domain of OsPRR37. Transcriptional analysis revealed that OsPRR37 suppressed Ghd7 expression in both ZH11 background under NLD conditions and the Zhenshan 97 background under natural short-day conditions. Consistently, the expression of Ehd1, Hd3a and RFT1 was enhanced by OsPRR37 in the ZH11 background. Genetic analysis indicated that the promotion of heading date and reduction in grain yield by OsPRR37 were partially dependent on Ghd7. Further investigation showed that the alternative function of OsPRR37 required an intact Ghd7-related regulatory pathway involving not only its upstream regulators OsGI and PhyB but also its interacting partner Hd1. Our study revealed the distinct role of OsPRR37 in the ZH11 background, which provides a more comprehensive understanding of OsPRR37 function and enriches the theoretical bases for improvement of rice heading date in the future.

scholarly journals Anthropometry, Carbohydrate and Lipid Metabolism in the East Flanders Prospective Twin Survey: Linkage of Candidate Genes Using Two Sib-Pair Based Variance Components Analyses

2008 ◽  
Vol 11 (5) ◽  
pp. 505-516 ◽  
Author(s):  
Nicole Y. Souren ◽  
Maurice P. Zeegers ◽  
Rob G. J. H. Janssen ◽  
Anja Steyls ◽  
Marij Gielen ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Birth Weight ◽  
Variance Components ◽  
Single Point ◽  
Hdl Cholesterol ◽  
Suggestive Linkage ◽  
Phenotypic Data ◽  
Linkage Analyses ◽  
Cholesterol Levels

AbstractInsulin resistance and obesity are underlying causes of type 2 diabetes and therefore much interest is focused on the potential genes involved. A series of anthropometric and metabolic characteristic were measured in 240 MZ and 112 DZ twin pairs recruited from the East Flanders Prospective Twin Survey. Microsatellite markers located close to ABCC8, ADIPOQ, GCK, IGF1, IGFBP1, INSR, LEP, LEPR, PPARγ and the RETN gene were genotyped. Univariate single point variance components linkage analyses were performed using two methods: (1) the standard method, only comprising the phenotypic and genotypic data of the DZ twin pairs and (2) the extended method, also incorporating the phenotypic data of the MZ twin pairs. Suggestive linkages (LOD > 1) were observed between the ABCC8 marker and waist-to-hip ratio and HDL-cholesterol levels. Both markers flanking ADIPOQ showed suggestive linkage with triglycerides levels, the upstream marker also with body mass and HDL-cholesterol levels. The IGFBP1 marker showed suggestive linkage with fat mass, fasting insulin and leptin levels and the LEP marker showed suggestive linkage with birth weight. This study suggests that DNA variants in ABCC8, ADIPOQ, IGFBP1 and LEP gene region may predispose to type 2 diabetes. In addition, the two methods used to perform linkage analyses yielded similar results. This was however not the case for birth weight where chorionicity seems to be an important confounder.

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 Involvement of a Nonstructural Protein in Poliovirus Capsid Assembly

2018 ◽  
Vol 93 (5) ◽  
Author(s):  
Oluwapelumi O. Adeyemi ◽  
Lee Sherry ◽  
Joseph C. Ward ◽  
Danielle M. Pierce ◽  
Morgan R. Herod ◽  
...  
Keyword(s):  
Early Stage ◽  
Selection Process ◽  
Nonstructural Protein ◽  
Infectious Clone ◽  
Regulatory Protein ◽  
Lethal Mutation ◽  
Site Directed Mutagenesis ◽  
Coding Region ◽  
Virion Assembly ◽  
Vp1 Protein

ABSTRACTVirus capsid proteins must perform a number of roles. These include self-assembly and maintaining stability under challenging environmental conditions, while retaining the conformational flexibility necessary to uncoat and deliver the viral genome into a host cell. Fulfilling these roles could place conflicting constraints on the innate abilities encoded within the protein sequences. In a previous study, we identified a number of mutations within the capsid-coding sequence of poliovirus (PV) that were established in the population during selection for greater thermostability by sequential treatment at progressively higher temperatures. Two mutations in the VP1 protein acquired at an early stage were maintained throughout this selection procedure. One of these mutations prevented virion assembly when introduced into a wild-type (wt) infectious clone. Here we show, by sequencing beyond the capsid-coding region of the heat-selected virions, that two mutations had arisen within the coding region of the 2A protease. Both mutations were maintained throughout the selection process. Introduction of these mutations into a wt infectious clone by site-directed mutagenesis considerably reduced replication. However, they permitted a low level of assembly of infectious virions containing the otherwise lethal mutation in VP1. The 2Apromutations were further shown to slow the kinetics of viral polyprotein processing, and we suggest that this delay improves the correct folding of the mutant capsid precursor protein to permit virion assembly.IMPORTANCERNA viruses, including poliovirus, evolve rapidly due to the error-prone nature of the polymerase enzymes involved in genome replication. Fixation of advantageous mutations may require the acquisition of complementary mutations which can act in concert to achieve a favorable phenotype. This study highlights a compensatory role of a nonstructural regulatory protein, 2Apro, for an otherwise lethal mutation of the structural VP1 protein to facilitate increased thermal resistance. Studying how viruses respond to selection pressures is important for understanding mechanisms which underpin emergence of resistance and could be applied to the future development of antiviral agents and vaccines.

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