RESUMEN
West Nile Virus (WNV) collected from 179 human blood donors in 25 US states and three Canadian provinces during the 2003 and 2004 epidemic seasons were genetically analyzed. The evolution of WNV during its Western spread was examined by envelope (E) gene sequencing of all 179 cases and full open reading frame sequencing of a subset of 20 WNV to determine if geographic and temporal segregation of distinct viral variants had occurred. Median joining network analysis was used to examine the genetic relationship between E gene variants and identified four large genetic clusters showing the gradual accumulation of mutations during the virus' western expansion. Two related WNV variants and their descendents, undetected in prior years, expanded in frequency. Apparent founder effects were observed in some regional outbreaks possibly due to local WNV colonization by a limited number of viruses. Amino acid mutations associated with newly expanding genetic variants reflect either selectively neutral mutational drift and/or mutations providing replicative advantages over the previously dominant forms of WNV.
Asunto(s)
Donantes de Sangre , Evolución Molecular , Filogenia , Fiebre del Nilo Occidental/epidemiología , Fiebre del Nilo Occidental/virología , Virus del Nilo Occidental/genética , Virus del Nilo Occidental/aislamiento & purificación , Análisis Mutacional de ADN , Productos del Gen env/genética , Genoma Viral/genética , Humanos , Datos de Secuencia Molecular , América del Norte/epidemiología , Sistemas de Lectura Abierta/genética , Reacción en Cadena de la Polimerasa , Factores de TiempoRESUMEN
BACKGROUND: Blood donation screening for West Nile virus (WNV) RNA by nucleic acid testing (NAT) was implemented in Canada in July 2003, and 14 WNV RNA-positive donations were identified. Samples were screened in minipools of six donations with a WNV assay (TaqScreen, Roche). Two of the donors were identified by single-donor screening that was initiated in the province of Saskatchewan, which had the highest prevalence of WNV in the country, in early September 2003. STUDY DESIGN AND METHODS: The original 14 samples and follow-up samples (2-35 days after donation), available from 13 of the 14 donors were tested with an in-house, real-time, quantitative WNV NAT assay that was specific for WNV. A Health Canada reference reagent was used for calibration. Immunoglobulin M (IgM) and immunoglobulin G (IgG) levels were determined with commercial enzyme-linked immunosorbent assay kits. RESULTS: All donors tested positive for the presence of WNV with the in-house assay. Two donors, 18 and 19, identified by single-donor testing, had extremely low levels of viremia and that could only be detected in 1:38 or 1:39 replicate tests. The titers of the remaining index samples ranged from below log2.8 (the limit of quantitation) to log4.7 NAT detectable units per mL. Three samples, from Donors 17, 18, and 19, were IgM-positive, whereas samples from Donors 18 and 19 were also IgG-positive. The remaining 10 donors with follow-up samples all seroconverted. CONCLUSION: The 14 WNV donor samples detected by routine screening were confirmed as WNV RNA-positive by a WNV RNA-specific in-house assay and by demonstration of seroconversion in 13 of the 14 donors.
Asunto(s)
Donantes de Sangre , Fiebre del Nilo Occidental/sangre , Fiebre del Nilo Occidental/epidemiología , Virus del Nilo Occidental/aislamiento & purificación , Anticuerpos Antivirales/sangre , Canadá/epidemiología , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Tamizaje Masivo , ARN Viral/análisis , Sensibilidad y Especificidad , Estudios Seroepidemiológicos , Fiebre del Nilo Occidental/inmunología , Virus del Nilo Occidental/genética , Virus del Nilo Occidental/inmunologíaRESUMEN
To date, no dominant mutation has been identified in a significant proportion of patients with type 1 von Willebrand disease (VWD). In this study, we examined 70 families as part of the Canadian Type 1 VWD Study. The entire VWF gene was sequenced for 1 index case, revealing 2 sequence variations: intron 30 (5312-19A>C) and exon 28 at Tyr1584Cys (4751A>G). The Tyr1584Cys variation was identified in 14.3% (10 of 70) of the families and was in phase with the 5312-19A>C variation in 7 (10.0%) families. Both variants were observed in 2 of 10 UK families with type 1 VWD, but neither variant was found in 200 and 100 healthy, unrelated persons, respectively. Mean von Willebrand factor antigen (VWF:Ag), VWF ristocetin cofactor (VWF:RCo), and factor VIII coagulant activity (FVIII:C) for the index cases in these families are 0.4 U/mL, 0.36 U/mL, and 0.54 U/mL, respectively, and VWF multimer patterns show no qualitative abnormalities. Aberrant VWF splicing was not observed in these patients, and both alleles of the VWF gene are expressed as RNA. Molecular dynamic simulation was performed on a homology model of the VWF-A2 domain containing the Tyr1584Cys mutation. This showed that no significant structural changes occur as a result of the substitution but that a new solvent-exposed reactive thiol group is apparent. Expression studies revealed that the Tyr1584Cys mutation results in increased intracellular retention of the VWF protein. We demonstrate that all the families with the Tyr1584Cys mutation share a common, evolved VWF haplotype, suggesting that this mutation is ancient. This is the first report of a mutation that segregates in a significant proportion of patients with type 1 VWD.
Asunto(s)
Sustitución de Aminoácidos , Efecto Fundador , Intrones/genética , Mutación Missense , Enfermedades de von Willebrand/genética , Factor de von Willebrand/genética , Alelos , Canadá/epidemiología , Codón/genética , Simulación por Computador , Evolución Molecular , Francia/epidemiología , Francia/etnología , Genotipo , Haplotipos , Humanos , Modelos Moleculares , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas/genética , Conformación Proteica , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Compuestos de Sulfhidrilo/análisis , Reino Unido/epidemiología , Reino Unido/etnología , Enfermedades de von Willebrand/epidemiología , Factor de von Willebrand/químicaRESUMEN
If gene therapy is to be an effective treatment modality for hemophilia A, therapeutic levels and tissue-restricted expression of factor VIII (FVIII) must be achieved through optimization of transgene expression. To this end, we incorporated three types of sequence elements into a canine B domain-deleted FVIII transgene cassette and individually evaluated their effect on FVIII transgene expression. Functional FVIII activity was initially assessed in vitro and hydrodynamic injection of the different transgene constructs into mice was subsequently used as a model to compare in vivo expression of the various modified transgenes. Our results demonstrate that in vitro transgene expression is, in these studies, not a good predictor of in vivo transgene performance. In vivo analysis of a hybrid tissue-restricted promoter element, consisting of a concatemer of five hepatocyte nuclear factor 1 (HNF-1) consensus-binding motifs juxtaposed to the human FVIII proximal promoter, indicates that it is as efficient at mediating expression of the FVIII protein as the cytomegalovirus promoter. Addition of the full-length canine FVIII 3'-UTR also enhances transgene expression of FVIII in vivo. Sequence analysis of the canine FVIII 3'-UTR and human FVIII 3'-UTR indicates that the former lacks instability sequences and may therefore be more effective in stabilizing FVIII mRNA. Subsequent inclusion of FVIII introns 16 and 17 into the natural locations of the transgene disrupted mRNA processing and abolished expression of the FVIII protein. Introduction of intron 17 proximal to the FVIII cDNA did not enhance in vivo expression of canine FVIII from the transgene.
Asunto(s)
Regiones no Traducidas 3'/genética , Factor VIII/genética , Terapia Genética , Hemofilia A/terapia , Factores de Transcripción/metabolismo , Animales , Perros , Factor VIII/metabolismo , Hemofilia A/genética , Intrones , Factores de Transcripción/genética , TransgenesRESUMEN
We have identified the causative mutation in the hemophilia A dog colony at Queen's University, Canada and have observed a striking similarity with the intron 22 inversion found in approximately 45% of severely affected hemophilia A patients. The canine hemophilia A phenotype arises from aberrant splicing and premature termination of transcription of the FVIII gene, resulting in a polyadenylated transcript lacking exons distal to 22 and terminating with a novel sequence element (NSE). In dogs and other species including humans, this NSE is present in low copy number. One copy of these sequences in the canine genome is within intron 22 and reveals differences in the hybridization banding patterns between normal and hemophilic DNA, suggestive of a large genomic rearrangement. The mutation mechanism may not be uncommon, as identical mutant transcripts were isolated from two hemophilia A littermates that are unrelated to the Queen's colony and from hemophiliac dogs in the colony at Chapel Hill.