RESUMEN
During June 2003, mosquito surveillance was conducted at a US Army installation and a US Military training site 2 km south of the demilitarized zone, Republic of Korea. Mosquitoes were collected using Mosquito Magnets™, sorted to species, and assayed for the presence of arboviruses. From the 3,149 mosquitoes that were sorted into 126 pools, one Aedes vexans nipponii pool (out of 73 pools) tested positive for flavivirus RNA by reverse transcription-PCR. After isolation from C6/36 cell culture supernatant, the viral genome was sequenced and found to be 98.9% related to Chaoyang virus, a potential arthropod-specific flavivirus. This report details the first identification of Chaoyang virus in the Republic of Korea and highlights its relationship to other flaviviruses.
Asunto(s)
Aedes/virología , Flavivirus/clasificación , Flavivirus/aislamiento & purificación , Insectos Vectores/virología , Aedes/clasificación , Animales , Secuencia de Bases , Línea Celular , Células Cultivadas , Flavivirus/genética , Genómica , Filogenia , Reacción en Cadena de la Polimerasa , ARN Viral/genética , ARN Viral/aislamiento & purificación , República de Corea , Análisis de Secuencia de ADN , Células VeroRESUMEN
The potential for genetic modification of biological warfare agents makes rapid identification of antibiotic resistant strains critical for the implementation of suitable infection control measures. The fluorinated quinolone, ciprofloxacin, is an antibiotic effective for treating bacterial infections by inhibiting the enzyme activity of the DNA type II topoisomerases DNA gyrase and topoisomerase IV. The genes that encode the subunits of DNA gyrase (gyrA and gyrB) and topo IV (par C and parE) contain hotspots within an area known as the quinolone resistance-determining region (QRDR). Base pair changes within this region give rise to mutations that cause resistance to the antibiotic by altering amino acids within the enzymes. Ciprofloxacin-resistant (cipro(r)) strains of Bacillus anthracis, Yersinia pestis, and Francisella tularensis with one or more known mutations within the QRDR of gyrA, gyrB, parC, and parE genes were tested with SimpleProbe and High Resolution Melt (HRM) dye chemistries and Pyrosequencing genetic analysis to evaluate the ability to rapidly detect ciprofloxacin-induced mutations. While SimpleProbe and Pyrosequencing successfully identified all known mutants, the HRM assay identified all but those resulting from G<-->C or A<-->T substitutions.
Asunto(s)
Bacillus anthracis/efectos de los fármacos , Ciprofloxacina/farmacología , Farmacorresistencia Bacteriana/genética , Francisella tularensis/efectos de los fármacos , Análisis de Secuencia de ADN/métodos , Yersinia pestis/efectos de los fármacos , Antiinfecciosos/farmacología , Bacillus anthracis/genética , Bacillus anthracis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Girasa de ADN/genética , Girasa de ADN/metabolismo , Topoisomerasa de ADN IV/genética , Topoisomerasa de ADN IV/metabolismo , Francisella tularensis/genética , Francisella tularensis/metabolismo , Pruebas de Sensibilidad Microbiana , Mutación , Reproducibilidad de los Resultados , Yersinia pestis/genética , Yersinia pestis/metabolismoRESUMEN
Identifying viral isolates from field-collected mosquitoes can be difficult and time-consuming, particularly in regions of the world where numerous closely related viruses are co-circulating (e.g., the Amazon Basin region of Peru). The use of molecular techniques may provide rapid and efficient methods for identifying these viruses in the laboratory. Therefore, we determined the complete nucleotide sequence of two South American eastern equine encephalomyelitis viruses (EEEVs): one member from the Peru-Brazil (Lineage II) clade and one member from the Argentina-Panama (Lineage III) clade. In addition, we determined the nucleotide sequence for the nonstructural P3 protein (nsP3) and envelope 2 (E2) protein genes of 36 additional isolates of EEEV from mosquitoes captured in Peru between 1996 and 2001. The 38 isolates were evenly distributed between lineages II and III virus groupings. However, analysis of the nsP3 gene for lineage III strongly suggested that the 19 isolates from this lineage could be divided into two sub-clades, designated as lineages III and IIIA. Compared with North American EEEV (lineage I, GA97 strain), we found that the length of the nsP3 gene was shorter in the strains isolated from South America. A total of 60 nucleotides was deleted in lineage II, 69 in lineage III, and 72 in lineage IIIA. On the basis of the sequences we determined for South American EEEVs and those for other viruses detected in the same area, we developed a series of primers for characterizing these viruses.
Asunto(s)
Culex/virología , Virus de la Encefalitis Equina del Este/genética , Animales , Virus de la Encefalitis Equina del Este/clasificación , Perú , Filogenia , Proteínas del Envoltorio Viral/genética , Proteínas no Estructurales Virales/genéticaRESUMEN
Eastern equine encephalitis (EEE) virus is a significant public health concern due to the high mortality rates observed in infected humans, equines and game birds. The EEE genomic sequences available prior to this report are based on laboratory strains with unknown passage histories that may contain an array of cell culture adaptations. Here we report the complete genomic sequences of two recently isolated EEE pathogenic strains with low passage histories. FL91-4697 was isolated in Florida from Aedes albopictus mosquitoes and GA97 was derived from brain tissue of a human fatality that occurred in 1997. Sequence alignment of these new genomes with the documented EEE's permitted us to generate a North American consensus sequence and identify regions of significant diversity. Sequence analysis of the FL91-4679 genome was essential to the production of an EEE infectious clone that is being used to create live attenuated vaccine candidates.
Asunto(s)
Virus de la Encefalitis Equina del Este/genética , Genoma Viral , Aedes/virología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/virología , Proteínas de la Cápside/genética , Secuencia de Consenso , Culicidae/virología , Virus de la Encefalitis Equina del Este/patogenicidad , Humanos , Datos de Secuencia Molecular , América del Norte , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Yersinia pestis, the causative agent of the zoonotic infection plague, is a major concern as a potential bioweapon. Current real-time PCR assays used for Y. pestis detection are based on plasmid targets, some of which may generate false-positive results. METHODS: Using the yp48 gene of Y. pestis, we designed and tested 2 real-time TaqMan minor groove binder (MGB) assays that allowed us to use chromosomal genes as both confirmatory and differential targets for Y. pestis. We also designed several additional assays using both Simple-Probe and MGB Eclipse probe technologies for the selective differentiation of Yersinia pseudotuberculosis from Y. pestis. These assays were designed around a 25-bp insertion site recently identified within the yp48 gene of Y. pseudotuberculosis. RESULTS: The Y. pestis-specific assay distinguished this bacterium from other Yersinia species but had unacceptable low-level detection of Y. pseudotuberculosis, a closely related species. Simple-Probe and MGB Eclipse probes specific for the 25-bp insertion detected only Y. pseudotuberculosis DNA. Probes that spanned the deletion site detected both Y. pestis and Y. pseudotuberculosis DNA, and the 2 species were clearly differentiated by a post-PCR melting temperature (Tm) analysis. The Simple-Probe assay produced an almost 7 degrees C Tm difference and the MGB Eclipse probe a slightly more than 4 degrees C difference. CONCLUSIONS: Our method clearly discriminates Y. pestis DNA from all other Yersinia species tested and from the closely related Y. pseudotuberculosis. These chromosomal assays are important both to verify the presence of Y. pestis based on a chromosomal target and to easily distinguish it from Y. pseudotuberculosis.
Asunto(s)
Cromosomas/genética , Marcación de Gen/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Análisis de Secuencia de ADN/métodos , Yersinia pestis/genética , Secuencia de Bases , ADN/genética , Datos de Secuencia Molecular , Sensibilidad y Especificidad , Temperatura de Transición , Yersinia pestis/clasificación , Yersinia pseudotuberculosis/clasificación , Yersinia pseudotuberculosis/genéticaRESUMEN
The mechanism by which arboviruses bypass the basal lamina of mosquito midgut cells and enter the body cavity has been unclear. Experiments using Venezuelan equine encephalitis viral replicon particles, which express the green fluorescent protein gene in cells, indicate the operation of tissue conduits, possibly involving tracheae and visceral muscles, that facilitate virus movement through the basal lamina. Ultrastructural studies of the midgut reveal evidence for possible complete penetration of the basal lamina by tracheal cells and regions of modified basal lamina associated with visceral muscle. The modified basal lamina closely resembles proventricular matrix material known to allow virus passage.
Asunto(s)
Arbovirus/aislamiento & purificación , Culicidae/virología , Sistema Digestivo/virología , Virus de la Encefalitis Equina Venezolana/aislamiento & purificación , Animales , Virus de la Encefalitis Equina Venezolana/genética , Genes Reporteros , Proteínas Fluorescentes Verdes , Corazón/virología , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Miocardio/ultraestructura , Replicón/genética , TransfecciónRESUMEN
In support of efforts to develop rapid diagnostic assays for use in the field, reverse transcription-polymerase chain reaction (RT-PCR) assays were developed to detect arboviruses circulating in the Amazon Basin region of Peru. Previous knowledge of arthropod/pathogen relationships allowed a focused evaluation to be conducted in November 2000 that assessed the feasibility and reliability of a mobile, rapid, field-expedient RT-PCR diagnostic system aimed at detecting eastern equine encephalitis virus (EEEV) in Culex (Melanoconion) pedroi mosquitoes. Modifications were made to a commercially available mobile molecular laboratory kit and assay procedures were tailored for use under harsh environmental conditions with field-collected and field-processed mosquitoes. From CO2 baited mosquito light traps, 3,227 Cx. (Mel.) pedroi mosquitoes were collected and sorted into 117 pools. The pools were processed and assayed in the field by RT-PCR and five of those pools were found positive for EEEV. Laboratory sequence analysis confirmed the presence of two distinct subtypes of EEEV.