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2.
Anal Bioanal Chem ; 409(14): 3657-3666, 2017 May.
Artículo en Inglés | MEDLINE | ID: mdl-28331957

RESUMEN

A new analytical method has been developed to detect three kinds of DNA simultaneously based on magnetic beads and color-encoded plasmonic nanocrystals. Magnetic beads modified with capture DNA are employed to collect the specific target DNA, and color-encoded plasmonic nanocrystals are applied to signal the target through DNA hybridization. As a proof of concept, three types of representative metal nanocrystals of gold nanoparticle (AuNP), gold nanorod (AuNR), and gold/silver nanoparticle (Au/AgNP) were employed to signal three dissimilar virus-related protective antigen genes, Ebola virus (EV), Variola virus (VV), and Bacillus anthracis (BA), respectively. Detection limits of 0.5-3 fM were obtained showing the high sensitivity for DNA detection. The microscopic discrimination of the encoded nanoparticles allows simple, rapid, accurate, and cost-effective detection of multiple DNA molecules, indicative of the potential in practical applications. Graphical abstract Development of a novel digital triplex DNA assay based on single-countable color-encoded plasmonic nanocrystals.


Asunto(s)
Técnicas Biosensibles/métodos , ADN Bacteriano/análisis , ADN Viral/análisis , Oro/química , Nanopartículas del Metal/química , Hibridación de Ácido Nucleico/métodos , Plata/química , Carbunco/microbiología , Bacillus anthracis/aislamiento & purificación , Sondas de ADN/química , Ebolavirus/aislamiento & purificación , Fiebre Hemorrágica Ebola/virología , Humanos , Ácidos Nucleicos Inmovilizados/química , Límite de Detección , Imanes/química , Nanopartículas del Metal/ultraestructura , Nanotubos/química , Nanotubos/ultraestructura , Viruela/virología , Virus de la Viruela/aislamiento & purificación
3.
J Virol Methods ; 236: 215-220, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27477914

RESUMEN

A method of one-stage rapid detection and differentiation of epidemiologically important variola virus (VARV), monkeypox virus (MPXV), and varicella-zoster virus (VZV) utilizing multiplex real-time TaqMan PCR assay was developed. Four hybridization probes with various fluorescent dyes and the corresponding fluorescence quenchers were simultaneously used for the assay. The hybridization probes specific for the VARV sequence contained FAM/BHQ1 as a dye/quencher pair; MPXV-specific, JOE/BHQ1; VZV-specific, TAMRA/BHQ2; and internal control-specific, Cy5/BHQ3. The specificity and sensitivity of the developed method were assessed by analyzing DNA of 32 strains belonging to orthopoxvirus and herpesvirus species.


Asunto(s)
Herpesvirus Humano 3/aislamiento & purificación , Monkeypox virus/aislamiento & purificación , Reacción en Cadena de la Polimerasa Multiplex/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Virus de la Viruela/aislamiento & purificación , Virología/métodos , Herpesvirus Humano 3/clasificación , Herpesvirus Humano 3/genética , Monkeypox virus/clasificación , Monkeypox virus/genética , Sensibilidad y Especificidad , Virus de la Viruela/clasificación , Virus de la Viruela/genética
4.
Adv Virus Res ; 95: 197-220, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27112283

RESUMEN

A historic review of the discovery of new viruses leads to reminders of traditions that have evolved over 118 years. One such tradition gives credit for the discovery of a virus to the investigator(s) who not only carried out the seminal experiments but also correctly interpreted the findings (within the technological context of the day). Early on, ultrafiltration played a unique role in "proving" that an infectious agent was a virus, as did a failure to find any microscopically visible agent, failure to show replication of the agent in the absence of viable cells, thermolability of the agent, and demonstration of a specific immune response to the agent so as to rule out duplicates and close variants. More difficult was "proving" that the new virus was the etiologic agent of the disease ("proof of causation")-for good reasons this matter has been revisited several times over the years as technologies and perspectives have changed. One tradition is that the discoverers get to name their discovery, their new virus (unless some grievous convention has been broken)-the stability of these virus names has been a way to honor the discoverer(s) over the long term. Several vignettes have been chosen to illustrate several difficulties in holding to the traditions (vignettes chosen include vaccinia and variola viruses, yellow fever virus, and influenza viruses. Crimean-Congo hemorrhagic fever virus, Murray Valley encephalitis virus, human immunodeficiency virus 1, Sin Nombre virus, and Ebola virus). Each suggests lessons for the future. One way to assure that discoveries are forever linked with discoverers would be a permanent archive in one of the universal virus databases that have been constructed for other purposes. However, no current database seems ideal-perhaps members of the global community of virologists will have an ideal solution.


Asunto(s)
Invenciones/historia , Ultrafiltración/historia , Virología/historia , Animales , Bases de Datos como Asunto , Ebolavirus/aislamiento & purificación , Ebolavirus/patogenicidad , Ebolavirus/fisiología , Virus de la Encefalitis del Valle Murray/aislamiento & purificación , Virus de la Encefalitis del Valle Murray/patogenicidad , Virus de la Encefalitis del Valle Murray/fisiología , VIH-1/aislamiento & purificación , VIH-1/patogenicidad , VIH-1/fisiología , Virus de la Fiebre Hemorrágica de Crimea-Congo/aislamiento & purificación , Virus de la Fiebre Hemorrágica de Crimea-Congo/patogenicidad , Virus de la Fiebre Hemorrágica de Crimea-Congo/fisiología , Historia del Siglo XIX , Historia del Siglo XX , Humanos , Orthomyxoviridae/aislamiento & purificación , Orthomyxoviridae/patogenicidad , Orthomyxoviridae/fisiología , Virus Sin Nombre/aislamiento & purificación , Virus Sin Nombre/patogenicidad , Virus Sin Nombre/fisiología , Ultrafiltración/estadística & datos numéricos , Virus Vaccinia/aislamiento & purificación , Virus Vaccinia/patogenicidad , Virus Vaccinia/fisiología , Virus de la Viruela/aislamiento & purificación , Virus de la Viruela/patogenicidad , Virus de la Viruela/fisiología , Recursos Humanos , Virus de la Fiebre Amarilla/aislamiento & purificación , Virus de la Fiebre Amarilla/patogenicidad , Virus de la Fiebre Amarilla/fisiología
5.
PLoS One ; 10(9): e0138484, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26381398

RESUMEN

CDC designated category A infectious agents pose a major risk to national security and require special action for public health preparedness. They include viruses that cause viral hemorrhagic fever (VHF) syndrome as well as variola virus, the agent of smallpox. VHF is characterized by hemorrhage and fever with multi-organ failure leading to high morbidity and mortality. Smallpox, a prior scourge, has been eradicated for decades, making it a particularly serious threat if released nefariously in the essentially non-immune world population. Early detection of the causative agents, and the ability to distinguish them from other pathogens, is essential to contain outbreaks, implement proper control measures, and prevent morbidity and mortality. We have developed a multiplex detection assay that uses several species-specific PCR primers to generate amplicons from multiple pathogens; these are then targeted in a ligase detection reaction (LDR). The resultant fluorescently-labeled ligation products are detected on a universal array enabling simultaneous identification of the pathogens. The assay was evaluated on 32 different isolates associated with VHF (ebolavirus, marburgvirus, Crimean Congo hemorrhagic fever virus, Lassa fever virus, Rift Valley fever virus, Dengue virus, and Yellow fever virus) as well as variola virus and vaccinia virus (the agent of smallpox and its vaccine strain, respectively). The assay was able to detect all viruses tested, including 8 sequences representative of different variola virus strains from the CDC repository. It does not cross react with other emerging zoonoses such as monkeypox virus or cowpox virus, or six flaviviruses tested (St. Louis encephalitis virus, Murray Valley encephalitis virus, Powassan virus, Tick-borne encephalitis virus, West Nile virus and Japanese encephalitis virus).


Asunto(s)
Fiebres Hemorrágicas Virales/diagnóstico , Reacción en Cadena de la Polimerasa Multiplex/métodos , Viruela/diagnóstico , Virus de la Viruela/aislamiento & purificación , Virus/aislamiento & purificación , Fiebres Hemorrágicas Virales/virología , Humanos , Viruela/virología
6.
PLoS One ; 10(7): e0131742, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26147658

RESUMEN

Although current nonhuman primate models of monkeypox and smallpox diseases provide some insight into disease pathogenesis, they require a high titer inoculum, use an unnatural route of infection, and/or do not accurately represent the entire disease course. This is a concern when developing smallpox and/or monkeypox countermeasures or trying to understand host pathogen relationships. In our studies, we altered half of the test system by using a New World nonhuman primate host, the common marmoset. Based on dose finding studies, we found that marmosets are susceptible to monkeypox virus infection, produce a high viremia, and have pathological features consistent with smallpox and monkeypox in humans. The low dose (48 plaque forming units) required to elicit a uniformly lethal disease and the extended incubation (preclinical signs) are unique features among nonhuman primate models utilizing monkeypox virus. The uniform lethality, hemorrhagic rash, high viremia, decrease in platelets, pathology, and abbreviated acute phase are reflective of early-type hemorrhagic smallpox.


Asunto(s)
Susceptibilidad a Enfermedades , Modelos Biológicos , Monkeypox virus/patogenicidad , Mpox/fisiopatología , Virus de la Viruela/patogenicidad , Animales , Callithrix , Chlorocebus aethiops , Masculino , Monkeypox virus/aislamiento & purificación , Estudios Prospectivos , Virus de la Viruela/aislamiento & purificación , Células Vero , Carga Viral
7.
J Clin Microbiol ; 53(4): 1406-10, 2015 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-25673790

RESUMEN

A public health response relies upon rapid and reliable confirmation of disease by diagnostic assays. Here, we detail the design and validation of two variola virus-specific real-time PCR assays, since previous assays cross-reacted with newly identified cowpox viruses. The assay specificity must continually be reassessed as other closely related viruses are identified.


Asunto(s)
Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Virus de la Viruela/aislamiento & purificación , Secuencia de Bases , ADN Viral/genética , Datos de Secuencia Molecular , Sensibilidad y Especificidad , Especificidad de la Especie
12.
Clin Microbiol Infect ; 20(3): 210-8, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24438205

RESUMEN

Smallpox is an infectious disease that is unique to humans, caused by a poxvirus. It is one of the most lethal of diseases; the virus variant Variola major has a mortality rate of 30%. People surviving this disease have life-long consequences, but also assured immunity. Historically, smallpox was recognized early in human populations. This led to prevention attempts--variolation, quarantine, and the isolation of infected subjects--until Jenner's discovery of the first steps of vaccination in the 18th century. After vaccination campaigns throughout the 19th and 20th centuries, the WHO declared the eradication of smallpox in 1980. With the development of microscopy techniques, the structural characterization of the virus began in the early 20th century. In 1990, the genomes of different smallpox viruses were determined; viruses could be classified in order to investigate their origin, diffusion, and evolution. To study the evolution and possible re-emergence of this viral pathogen, however, researchers can only use viral genomes collected during the 20th century. Cases of smallpox in ancient periods are sometimes well documented, so palaeomicrobiology and, more precisely, the study of ancient smallpox viral strains could be an exceptional opportunity. The analysis of poxvirus fragmented genomes could give new insights into the genetic evolution of the poxvirus. Recently, small fragments of the poxvirus genome were detected. With the genetic information obtained, a new phylogeny of smallpox virus was described. The interest in conducting studies on ancient strains is discussed, in order to explore the natural history of this disease.


Asunto(s)
Viruela/historia , Animales , Evolución Biológica , Historia del Siglo XVI , Historia del Siglo XVII , Historia del Siglo XVIII , Historia del Siglo XIX , Historia del Siglo XX , Humanos , Viruela/diagnóstico , Viruela/epidemiología , Viruela/virología , Virus de la Viruela/genética , Virus de la Viruela/aislamiento & purificación
13.
Emerg Infect Dis ; 20(2): 177-84, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24447382

RESUMEN

Although it has been >30 years since the eradication of smallpox, the unearthing of well-preserved tissue material in which the virus may reside has called into question the viability of variola virus decades or centuries after its original occurrence. Experimental data to address the long-term stability and viability of the virus are limited. There are several instances of well-preserved corpses and tissues that have been examined for poxvirus viability and viral DNA. These historical specimens cause concern for potential exposures, and each situation should be approached cautiously and independently with the available information. Nevertheless, these specimens provide information on the history of a major disease and vaccination against it.


Asunto(s)
ADN Viral/aislamiento & purificación , Viabilidad Microbiana , Viruela/prevención & control , Viruela/virología , Virus de la Viruela/fisiología , Autopsia , Cadáver , Historia del Siglo XVIII , Historia del Siglo XIX , Historia del Siglo XX , Historia Antigua , Humanos , Viruela/historia , Viruela/transmisión , Vacuna contra Viruela/inmunología , Manejo de Especímenes/métodos , Vacunación , Virus Vaccinia/inmunología , Virus de la Viruela/aislamiento & purificación , Virus de la Viruela/patogenicidad
16.
Klin Lab Diagn ; (12): 39-42, 2011 Dec.
Artículo en Ruso | MEDLINE | ID: mdl-22416430

RESUMEN

The set of specific oligonucleotide primers and hybridization testers with the support of TaqMan in real time is proposed to identify and differentiate such human pathogenic orthopoxviruses as smallpox virus, monkey smallpox virus, cowpox virus, variolovaccine. The analytical specificity of the set is determined upon the example of 20 strains of 6 types of human pathogenic orthopoxviruses and made up 100%. The sensibility is established using recombinant plasmids containing orthopoxviruses sequence. The minimal revealed amount of plasmid made up to 20 copies for smallpox and monkey smallpox viruses, 10 copies for cowpox and 60 copies for variolovaccine in reaction.


Asunto(s)
ADN Viral/análisis , Orthopoxvirus/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Infecciones por Poxviridae/diagnóstico , Secuencia de Aminoácidos , Animales , Virus de la Viruela Vacuna/aislamiento & purificación , Cartilla de ADN/química , ADN Viral/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Infecciones por Poxviridae/virología , Sensibilidad y Especificidad , Virus Vaccinia/aislamiento & purificación , Virus de la Viruela/aislamiento & purificación
18.
Mol Gen Mikrobiol Virusol ; (1): 29-32, 2010.
Artículo en Ruso | MEDLINE | ID: mdl-20361666

RESUMEN

Comparative analysis of amino acid and nucleotides sequences of ORFs located in extended segments of the terminal variable regions in variola virus genome detected a promising locus for viral genotyping according to the geographic origin. This is ORF O1L of VARV. The primers were calculated for synthesis of this ORF fragment by PCR, which makes it possible to distinguish South America-Western Africa genotype from other VARV strains. Subsequent RFLP analysis reliably differentiated Asian strains from African strains (except Western Africa isolates). This method has been tested using 16 VARV strains from various geographic regions. The developed approach is simple, fast and reliable.


Asunto(s)
Reacción en Cadena de la Polimerasa/métodos , Viruela/virología , Virus de la Viruela/clasificación , Virus de la Viruela/aislamiento & purificación , África , Secuencia de Aminoácidos , Repetición de Anquirina/genética , Asia , Cartilla de ADN/genética , Variación Genética , Genoma Viral/genética , Humanos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Polimorfismo de Longitud del Fragmento de Restricción , Sensibilidad y Especificidad , Alineación de Secuencia , América del Sur , Virus de la Viruela/genética
19.
Vopr Virusol ; 54(6): 28-33, 2009.
Artículo en Ruso | MEDLINE | ID: mdl-20030279

RESUMEN

A kit of specific oligonucleotide primers and hybridization probes has been proposed to detect orthopoxviruses (OPV) and to discriminate human pathogenic viruses, such as variola virus and monkey virus by real-time polymerase chain reaction (PCR). For real-time PCR, the following pairs of fluorophore and a fluorescence quencher were used: TAMRA-BHQ2 for genus-specific probes and FAM-BHQ1 for species-specific ones (variola virus, monkeypox virus, ectomelia virus). The specificity of this assay was tested on 38 strains of 6 OPV species and it was 100%.


Asunto(s)
Monkeypox virus/aislamiento & purificación , Mpox/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Viruela/diagnóstico , Virus de la Viruela/aislamiento & purificación , Animales , Cartilla de ADN/genética , ADN Viral/análisis , Humanos , Mpox/genética , Monkeypox virus/genética , Viruela/genética , Virus de la Viruela/genética
20.
J Gen Virol ; 90(Pt 4): 792-798, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19264626

RESUMEN

Smallpox (infection with Orthopoxvirus variola) remains a feared illness more than 25 years after its eradication. Historically, case-fatality rates (CFRs) varied between outbreaks (<1 to approximately 40 %), the reasons for which are incompletely understood. The extracellular enveloped virus (EEV) form of orthopoxvirus progeny is hypothesized to disseminate infection. Investigations with the closely related Orthopoxvirus vaccinia have associated increased comet formation (EEV production) with increased mouse mortality (pathogenicity). Other vaccinia virus genetic manipulations which affect EEV production inconsistently support this association. However, antisera against vaccinia virus envelope protect mice from lethal challenge, further supporting a critical role for EEV in pathogenicity. Here, we show that the increased comet formation phenotypes of a diverse collection of variola viruses associate with strain phylogeny and geographical origin, but not with increased outbreak-related CFRs; within clades, there may be an association of plaque size with CFR. The mechanisms for variola virus pathogenicity probably involves multiple host and pathogen factors.


Asunto(s)
Brotes de Enfermedades , Filogenia , Viruela/mortalidad , Virus de la Viruela/crecimiento & desarrollo , Virus de la Viruela/patogenicidad , Ensayo de Placa Viral , Animales , Línea Celular , Humanos , Ratones , Fenotipo , Conejos , Viruela/epidemiología , Viruela/virología , Tasa de Supervivencia , Virus de la Viruela/genética , Virus de la Viruela/aislamiento & purificación , Proteínas del Envoltorio Viral/metabolismo , Virulencia
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