RESUMO
BACKGROUND: The emergence of Zika virus (ZIKV) in 2015 to 2016 created a global public health crisis and an urgent need for accurate detection assays. Nucleic acid testing (NAT) is the most specific and sensitive technology for early detection of ZIKV. Various NAT protocols have been created, but until recently, assessment of assay performance and comparative studies were hampered by the lack of available standards and reference reagents. STUDY DESIGN AND METHODS: The Center for Biologics Evaluation and Research/Food and Drug Administration responded to this crisis with the generation of two ZIKV-RNA reference reagents (ZIKV-RRs) for use in the development, validation, and assessment of performance of ZIKV-NAT assays. These reagents were produced from heat-inactivated (HI) ZIKV culture supernatant stock from two strains (PRVABC59 and FSS13025) diluted in dialyzed, defibrinated human plasma and lyophilized for evaluation in collaborative studies. The liquid, HI stock had been shared with the Paul-Ehrlich-Institute (Germany) and were included in the collaborative validation studies for the World Health Organization International Standard for ZIKV (WHO ZIKV IS). RESULTS: NAT-detectable units (NDUs)/mL were determined in a collaborative study that led to the assignment of 5.77 log NDUs/mL for PRVABC59 and 5.54 log NDUs/mL for FSS13025 as the final concentrations of the FDA ZIKV-RRs. CONCLUSION: We have established well-characterized reference reagents for ZIKV to facilitate evaluation of existing NAT assays and development of novel ZIKV assays which are correlated to that of the First WHO ZIKV IS. Vials of the ZIKV-RRs are available to qualified organizations upon request.
Assuntos
Surtos de Doenças , Técnicas de Amplificação de Ácido Nucleico/normas , RNA Viral/normas , Infecção por Zika virus/epidemiologia , Zika virus/genética , Animais , Chlorocebus aethiops , Criopreservação , Liofilização , Humanos , Indicadores e Reagentes , Técnicas de Amplificação de Ácido Nucleico/métodos , Saúde Pública , Estabilidade de RNA , RNA Viral/sangue , RNA Viral/isolamento & purificação , Padrões de Referência , Sensibilidade e Especificidade , Células Vero , Viremia/sangue , Viremia/diagnóstico , Inativação de Vírus , Organização Mundial da Saúde , Zika virus/isolamento & purificação , Infecção por Zika virus/sangue , Infecção por Zika virus/diagnósticoRESUMO
BACKGROUND: Dengue is the most important reemerging mosquito-borne viral disease worldwide. Caused by dengue virus (DENV), a member of the genus Flavivirus in the Flaviviridae family, dengue can be asymptomatic (approx. 80% of cases) or symptomatic, ranging from a flu-like illness known as dengue fever, to a life-threatening form called severe dengue. DENV is primarily transmitted from human to human through the bite of mosquitoes of the genus Aedes; however, it is also transmissible by transfusion of blood and blood components and by solid organ transplant. Nucleic acid test (NAT) assays are considered the most appropriate approach for blood donor screening for recent DENV infections, but there is no Food and Drug Administration-approved assay for the screening of blood for DENV. STUDY DESIGN AND METHODS: An international collaborative study was conducted to assess the suitability of reference reagent (RR) candidates for DENV Types 1 to 4 RNA for use in NAT-based assays. RESULTS: Two sets of RR candidates were prepared for each DENV type, one liquid frozen (Set 1) and one lyophilized (Set 2). A total of 28 laboratories from 20 countries agreed to participate in the study, of which 21 submitted the results for qualitative and/or quantitative assessments. CONCLUSION: The World Health Organization has established the lyophilized materials as international RRs for DENV RNA with a unitage of 13,500, 69,200, 23,400, and 33,900 units/mL for DENV-1 to -4, respectively.
Assuntos
Segurança do Sangue/normas , Vírus da Dengue/genética , RNA Viral/sangue , Análise de Sequência de RNA/normas , Doadores de Sangue , Liofilização , Congelamento , Humanos , Indicadores e Reagentes/normas , Cooperação Internacional , RNA Viral/classificação , RNA Viral/isolamento & purificação , Análise de Sequência de RNA/métodos , Reação Transfusional , Organização Mundial da SaúdeAssuntos
Negro ou Afro-Americano/genética , Polimorfismo de Nucleotídeo Único , Sistema do Grupo Sanguíneo Rh-Hr/metabolismo , Alelos , População Negra/genética , Doadores de Sangue , Estudos de Coortes , Éxons/genética , Humanos , Fenótipo , Sistema do Grupo Sanguíneo Rh-Hr/genética , Sistema do Grupo Sanguíneo Rh-Hr/imunologia , Mutação Silenciosa/genéticaRESUMO
Patients who carry Rhesus (RH) blood group variants may develop Rh alloantibodies requiring matched red blood cell transfusions. Serologic reagents for Rh variants often fail to specifically identify variant Rh antigens and are in limited supply. Therefore, red blood cell genotyping assays are essential for managing transfusions in patients with clinically relevant Rh variants. Well-characterized DNA reference reagents are needed to ensure quality and accuracy of the molecular tests. Eight lyophilized DNA reference reagents, representing 21 polymorphisms in RHD and RHCE, were produced from an existing repository of immortalized B-lymphoblastoid cell lines at the Center for Biologics Evaluation and Research/US Food and Drug Administration. The material was validated through an international collaborative study involving 17 laboratories that evaluated each DNA candidate using molecular assays to characterize RHD and RHCE alleles, including commercial platforms and laboratory-developed testing, such as Sanger sequencing, next-generation sequencing, and third-generation sequencing. The genotyping results showed 99.4% agreement with the expected results for the target RH polymorphisms and 87.9% for RH allele agreement. Most of the discordant RH alleles results were explained by a limited polymorphism coverage in some genotyping methods. Results of stability and accelerated degradation studies support the suitability of these reagents for use as reference standards. The collaborative study results demonstrate the qualification of these eight DNA reagents for use as reference standards for RH blood group genotyping assay development and analytical validation.
Assuntos
Técnicas de Genotipagem , Sistema do Grupo Sanguíneo Rh-Hr , Humanos , Sistema do Grupo Sanguíneo Rh-Hr/genética , Técnicas de Genotipagem/métodos , Técnicas de Genotipagem/normas , Genótipo , Alelos , DNA/genética , Padrões de Referência , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Polimorfismo Genético , Indicadores e ReagentesRESUMO
Rio Bravo virus (RBV) is a member of the family Flaviviridae, genus Flavivirus. It belongs to a group of viruses in the genus with no known vector. In this report, we analyze the complete genome of the prototype RBV, strain M64.
Assuntos
Flavivirus/genética , Genoma Viral , Humanos , Dados de Sequência MolecularRESUMO
Zika virus (ZIKV) emerged in the Americas in 2015, presenting unique challenges to public health. Unlike other arboviruses of the Flaviviridae family, it is transmissible by sexual contact, which facilitates the spread of the virus into new geographic areas. Additionally, ZIKV can be transmitted from mother to fetus, causing microcephaly and other severe developmental abnormalities. Reliable and easy-to-work-with clones of ZIKV expressing heterologous genes will significantly facilitate studies aimed at understanding the virus pathogenesis and tissue tropism. Here, we developed and characterized two novel approaches for expression of heterologous genes of interest in the context of full-length ZIKV genome and compared them to two previously published strategies for ZIKV-mediated gene expression. We demonstrated that among the four tested viruses expressing nLuc gene, the virus constructed using a newly developed approach of partial capsid gene duplication (PCGD) attained the highest titer in Vero cells and resulted in the highest level of nLuc expression. Suitability of the PCGD approach for expression of different genes of interest was validated by replacing nLuc sequence with that of eGFP gene. The generated constructs were further characterized in cell culture. Potential applications of ZIKV clones stably expressing heterologous genes include development of detection assays, antivirals, therapeutics, live imaging systems, and vaccines.
Assuntos
Genética Reversa/métodos , Zika virus/genética , Animais , Antígenos Heterófilos/genética , Proteínas do Capsídeo/genética , Chlorocebus aethiops , Clonagem Molecular , Elementos de DNA Transponíveis , Expressão Gênica , Genes Virais , Genoma Viral , Proteínas de Fluorescência Verde/genética , Humanos , Células Vero , Replicação ViralRESUMO
The glycoprotein encoded by the ACKR1 gene expresses the Duffy blood group antigens and is a receptor for malaria parasites. We recently described 18 long-range ACKR1 alleles in an autochthonous population of a malaria endemic region. Extending this work, we sequenced the gene in a 53-sample repository established by the US Food and Drug Administration (FDA) as reference reagents for blood group genotyping. The FDA samples have been characterized for 19 genes; however, long-range haplotype information for these genes, including ACKR1, was lacking. We used a hybrid approach, novel for this type of gene, to characterize ACKR1 by combining two next-generation sequencing technologies, the short-read massively parallel sequencing and the long-read nanopore sequencing. The expedient integration of data from both next-generation sequencing systems were necessary and sufficient to allow determination of all 25 long-range ACKR1 alleles found in the 53 samples accurately. All 25 alleles identified in our current FDA cohort were novel and, unexpectedly, none had been observed among the 18 alleles in our previous study. The alleles will be useful for validation, calibration, and proficiency testing of red cell genotyping. The lack of any overlap between the ACKR1 alleles in the two studies documents differences in mutation rate and recombination frequency among populations. The exact haplotype and their interethnic or interpopulation dissimilarities can influence disease susceptibility and therapy.
Assuntos
Alelos , Pareamento de Bases/genética , Antígenos de Grupos Sanguíneos/genética , Sistema do Grupo Sanguíneo Duffy/genética , Técnicas de Genotipagem/normas , Receptores de Superfície Celular/genética , United States Food and Drug Administration , Sequência de Bases , Etiópia , Humanos , Polimorfismo de Nucleotídeo Único/genética , Padrões de Referência , Estados UnidosRESUMO
Zika virus (ZIKV) caused a public health threat in the United States in 2016, leading to rapid development and implementation of blood screening assays for ZIKV RNA. Several ZIKV sequences from clinical cases have been reported, but none from asymptomatic/pre-symptomatic infections. We isolated and sequenced ZIKV from asymptomatic/pre-symptomatic blood donor (ABD-ZIKV) samples and compared with reported clinical sequences. Twelve ABD-ZIKV isolates were produced from 67 cultivated samples, and isolates were genetically similar among themselves. Most isolates shared mutations with the clinical isolate PRVABC59 2015, whereas two ABD-ZIKV isolates shared specific mutations with U.S. clinical isolates from 2016. The ABD-ZIKV strains clustered into two distinct subclades: one comprised mostly ABD-ZIKV from Puerto Rico, and another one comprised ABD-ZIKV from Florida and QTX-02 isolate (Puerto Rico). In this study, we showed the circulation of two slightly distinct virus strains among Puerto Rico blood donors, one of which was also reported in Florida.
Assuntos
Doadores de Sangue , Filogenia , Infecção por Zika virus/sangue , Zika virus/genética , Zika virus/isolamento & purificação , Adulto , Doenças Transmissíveis Emergentes/sangue , Doenças Transmissíveis Emergentes/epidemiologia , Florida/epidemiologia , Genômica , Humanos , Porto Rico/epidemiologia , Infecção por Zika virus/diagnóstico , Infecção por Zika virus/epidemiologiaRESUMO
Extended blood group genotyping is an invaluable tool used for prevention of alloimmunization. Genotyping is particularly suitable when antigens are weak, specific antisera are unavailable, or accurate phenotyping is problematic because of a disease state or recent transfusions. In addition, genotyping facilitates establishment of mass-scale patient-matched donor databases. However, standardization of genotyping technologies has been hindered by the lack of reference panels. A well-characterized renewable reference panel for standardization of blood group genotyping was developed. The panel consists of genomic DNA lyophilized and stored in glass vials. Genomic DNA was extracted in bulk from immortalized lymphoblastoid cell lines, generated by Epstein-Barr virus transformation of peripheral blood lymphocytes harvested from volunteer blood donors. The panel was validated by an international collaborative study involving 28 laboratories that tested each DNA panel member for 41 polymorphisms associated with 17 blood group systems. Overall, analysis of genotyping results showed >98% agreement with the expected outcomes, demonstrating suitability of the material for use as reference. Highest levels of discordance were observed for the genes CR1, CD55, BSG, and RHD. Although limited, observed inconsistencies and procedural limitations reinforce the importance of reference reagents to standardize and harmonize results. Results of stability and accelerated degradation studies support the suitability of this panel for use as reference reagent for blood group genotyping assay development and standardization.
Assuntos
Antígenos de Grupos Sanguíneos/genética , DNA/genética , Genoma Humano , Técnicas de Genotipagem/normas , Alelos , Linhagem Celular , Eritrócitos/metabolismo , Genótipo , Humanos , Fenótipo , Polimorfismo Genético , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
We report here the complete genome sequences of two Zika virus strains (FSS13025 and PRVABC59) used for formulation of CBER/FDA RNA reference reagents and lot release panels for use with nucleic acid technology (NAT) testing.
RESUMO
West Nile virus (WNV) has become endemic in the Western Hemisphere since its first introduction in the United States in 1999. An important factor associated with annual reoccurrence of WNV outbreaks in the U.S. is viral adaptation to domestic mosquitoes and birds through accumulation of spontaneous mutations in the WNV genome. Newly emerged mutations in the viral genome can potentially negatively affect the performance of existing diagnostic and screening assays and future vaccines. Therefore, the genetic monitoring of the WNV viral population during annual outbreaks is extremely important for public health and can only be achieved by application of efficient sample preparation methods followed by high throughput genetic analysis. In this study, we developed and evaluated a method for specific isolation of WNV genomic RNA from plasma samples without cultivation of the virus in cells. In combination with the microarray-based genetic analysis of the isolated WNV genomic RNA, this approach is suitable for fast, high throughput genotyping of circulating WNV genetic variants. The methods were evaluated using WNV isolates from the 1999-2012U.S. epidemics.
Assuntos
Variação Genética , Genoma Viral , Análise em Microsséries/métodos , RNA Viral/sangue , RNA Viral/isolamento & purificação , Vírus do Nilo Ocidental/genética , Animais , Aves/virologia , Culicidae/virologia , Epidemias , Genótipo , Mutação , RNA Viral/genética , Estados Unidos/epidemiologia , Febre do Nilo Ocidental/epidemiologia , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/isolamento & purificaçãoRESUMO
The recent emergence of Zika virus underscores the need for new strategies for a rapid development of safe flavivirus vaccines. Using another flavivirus (Langat virus [LGTV]) that belongs to the group of tick-borne flaviviruses as a model, we describe a dual strategy for virus attenuation which synergistically accesses the specificity of microRNA (miRNA) genome targeting and the effectiveness of internal ribosome entry site (IRES) insertion. To increase the stability and immunogenicity of bicistronic LGTVs, we developed a novel approach in which the capsid (C) protein gene was relocated into the 3' noncoding region (NCR) and expressed under translational control from an IRES. Engineered bicistronic LGTVs carrying multiple target sequences for brain-specific miRNAs were stable in Vero cells and induced adaptive immunity in mice. Importantly, miRNA-targeted bicistronic LGTVs were not pathogenic for either newborn mice after intracranial inoculation or adult immunocompromised mice (SCID or type I interferon receptor knockout) after intraperitoneal injection. Moreover, bicistronic LGTVs were restricted for replication in tick-derived cells, suggesting an interruption of viral transmission in nature by arthropod vectors. This approach is suitable for reliable attenuation of many flaviviruses and may enable development of live attenuated flavivirus vaccines.IMPORTANCE The recent emergence of Zika virus underscores the need for new strategies for a rapid development of safe flavivirus vaccines. Allied separately attenuating approaches based on (i) microRNA genome targeting and (ii) internal ribosome entry site insertion are not sufficient for relievable attenuation of neurotropic flavivirus pathogenesis. Here, we describe a novel dual strategy that combines the specificity of miRNA-based and the effectiveness of IRES-based attenuating approaches, allowing us to overcome these critical limitations. This developed approach provides a robust platform for reliable attenuation of many flaviviruses and may enable development of live flavivirus vaccines.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos/genética , Vírus da Encefalite Transmitidos por Carrapatos/imunologia , Regulação da Expressão Gênica , Sítios Internos de Entrada Ribossomal , MicroRNAs , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Modelos Animais de Doenças , Vírus da Encefalite Transmitidos por Carrapatos/patogenicidade , Vírus da Encefalite Transmitidos por Carrapatos/fisiologia , Encefalite Transmitida por Carrapatos/imunologia , Encefalite Transmitida por Carrapatos/patologia , Encefalite Transmitida por Carrapatos/prevenção & controle , Encefalite Transmitida por Carrapatos/virologia , Expressão Gênica , Rearranjo Gênico , Camundongos , Recombinação Genética , Análise de Sobrevida , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/isolamento & purificação , Células Vero , Carga Viral , Vacinas Virais/administração & dosagem , Vacinas Virais/isolamento & purificação , Virulência , Replicação ViralRESUMO
Dengue virus (DENV), a member of the Flaviviridae family, is the most common and clinically significant arbovirus in the world and is endemic in more than 100 countries. Here, we report the complete sequences of four DENV serotypes used in the development of the CBER/FDA RNA reference reagents and WHO International Standard candidates for nucleic acid testing.
RESUMO
A series of explorative cross-coupling reactions have been developed to investigate the local nanoscale environment around catalytically active Pd(ii)complexes encapsulated within hollow graphitised nanofibers (GNF). Two new fullerene-containing and fullerene-free Pd(ii)Salen catalysts have been synthesised, and their activity and selectivity towards different substrates has been explored in nanoreactors. The catalysts not only show a significant increase in activity and stability upon heterogenisation at the graphitic step-edges inside the GNF channel, but also exhibit a change in selectivity affected by the confinement which alters the distribution of isomeric products of the reaction. Furthermore, the observed selectivity changes reveal unprecedented details regarding the location and orientation of the catalyst molecules inside the GNF nanoreactor, inaccessible by any spectroscopic or microscopic techniques, thus shedding light on the precise reaction environment inside the molecular catalyst-GNF nanoreactor.
RESUMO
West Nile virus (WNV) is an arbovirus maintained in nature in a bird-mosquito enzootic cycle which can also infect other vertebrates including humans. WNV is now endemic in the United States (U.S.), causing yearly outbreaks that have resulted in an estimated total of 4-5 million human infections. Over 41,700 cases of West Nile disease, including 18,810 neuroinvasive cases and 1,765 deaths, were reported to the CDC between 1999 and 2014. In 2012, the second largest West Nile outbreak in the U.S. was reported, which caused 5,674 cases and 286 deaths. WNV continues to evolve, and three major WNV lineage I genotypes (NY99, WN02, and SW/WN03) have been described in the U.S. since introduction of the virus in 1999. We report here the WNV sequences obtained from 19 human samples acquired during the 2012 U.S. outbreak and our examination of the evolutionary dynamics in WNV isolates sequenced from 1999-2012. Maximum-likelihood and Bayesian methods were used to perform the phylogenetic analyses. Selection pressure analyses were performed with the HyPhy package using the Datamonkey web-server. Using different codon-based and branch-site selection models, we detected a number of codons subjected to positive pressure in WNV genes. Thirteen of the 19 completely sequenced isolates from 10 U.S. states were genetically similar, sharing up to 55 nucleotide mutations and 4 amino acid substitutions when compared with the prototype isolate WN-NY99. Overall, these analyses showed that following a brief contraction in 2008-2009, WNV genetic divergence in the U.S. continued to increase in 2012, and that closely related variants were found across a broad geographic range of the U.S., coincident with the second-largest WNV outbreak in U.S.
Assuntos
Doadores de Sangue , Epidemias , Variação Genética , Febre do Nilo Ocidental/epidemiologia , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/genética , Substituição de Aminoácidos , Sequência de Bases , Teorema de Bayes , Genótipo , Humanos , Mutação , Filogenia , Análise de Sequência de DNA , Estados Unidos/epidemiologia , Vírus do Nilo Ocidental/classificação , Vírus do Nilo Ocidental/isolamento & purificaçãoRESUMO
Since its initial isolation in Uganda in 1937 through the present, West Nile virus (WNV) has become an important cause of human and animal disease worldwide. WNV, an enveloped virus of the genus Flavivirus, is naturally maintained in an enzootic cycle between birds and mosquitoes, with occasional epizootic spillover causing disease in humans and horses. The mosquito vectors for WNV are widely distributed worldwide, and the known geographic range of WNV transmission and disease has continued to increase over the past 77 years. While most human infections with WNV are asymptomatic, severe neurological disease may develop resulting in long-term sequelae or death. Surveillance and preventive measures are an ongoing need to reduce the public health impact of WNV in areas with the potential for transmission.
Assuntos
Culicidae/fisiologia , Insetos Vetores/fisiologia , Febre do Nilo Ocidental/epidemiologia , Febre do Nilo Ocidental/transmissão , Vírus do Nilo Ocidental/fisiologia , Animais , Culicidae/virologia , Humanos , Insetos Vetores/virologiaRESUMO
A computational approach for identification and assessment of genomic sequence variability (GeneSV) is described. For a given nucleotide sequence, GeneSV collects information about the permissible nucleotide variability (changes that potentially preserve function) observed in corresponding regions in genomic sequences, and combines it with conservation/variability results from protein sequence and structure-based analyses of evaluated protein coding regions. GeneSV was used to predict effects (functional vs. non-functional) of 37 amino acid substitutions on the NS5 polymerase (RdRp) of dengue virus type 2 (DENV-2), 36 of which are not observed in any publicly available DENV-2 sequence. 32 novel mutants with single amino acid substitutions in the RdRp were generated using a DENV-2 reverse genetics system. In 81% (26 of 32) of predictions tested, GeneSV correctly predicted viability of introduced mutations. In 4 of 5 (80%) mutants with double amino acid substitutions proximal in structure to one another GeneSV was also correct in its predictions. Predictive capabilities of the developed system were illustrated on dengue RNA virus, but described in the manuscript a general approach to characterize real or theoretically possible variations in genomic and protein sequences can be applied to any organism.
RESUMO
During 2010 and 2011, the Loreto region of Peru experienced a dengue outbreak of unprecedented magnitude and severity for the region. This outbreak coincided with the reappearance of dengue virus-2 (DENV-2) in Loreto after almost 8 years. Whole-genome sequence indicated that DENV-2 from the outbreak belonged to lineage II of the southeast Asian/American genotype and was most closely related to viruses circulating in Brazil during 2007 and 2008, whereas DENV-2 previously circulating in Loreto grouped with lineage I (DENV-2 strains circulating in South America since 1990). One amino acid substitution (NS5 A811V) in the 2010 and 2011 isolates resulted from positive selection. However, the 2010 and 2011 DENV-2 did not replicate to higher titers in monocyte-derived dendritic cells and did not infect or disseminate in a higher proportion of Aedes aegypti than DENV-2 isolates previously circulating in Loreto. These results suggest that factors other than enhanced viral replication played a role in the severity of this outbreak.