RESUMO
Pyrethroids are extensively used to control adult populations of the arboviral vector Aedes aegypti, raising concerns regarding the increasing frequency and distribution of insecticide resistance mutations (kdr: knock-down resistance) in the voltage-gated sodium channel gene (Nav). The widespread use of pyrethroids imposes a threat to the success of mosquito control and the environment. In this study, we investigated the presence of two kdr mutations (V1016I and F1534C) in the Nav gene and their distribution across four neighborhoods in Posadas, Argentina, with different Ae. aegypti abundance and contrasting socioeconomic status (SES). Alleles at each locus were interrogated using TaqMan SNP genotyping assays in DNA extracted from adult females collected in a longitudinal study. We report the presence of both pyrethroid resistance alleles (kdr 1016I = 29.08%; kdr 1534C = 70.70%) among adult females. The frequency of combined kdr genotypes reveals that approximately 70% of local adult females have enhanced resistance to pyrethroids. Both, the proportion of resistant adult females (with at least one kdr allele in each locus) and Ae. aegypti abundance showed an uneven distribution between neighborhoods with different SES (p < 0.001). In high-SES neighborhoods, we found more mosquitoes and a higher frequency of pyrethroid resistance, possibly as a consequence of different public health interventions, social habits, and insecticide use. This is the first report of kdr mutations in Ae. Aegypti in the northeast region of Argentina. Our results focus on the need for within-population (city) distribution analyses of kdr mutations and highlight the relevance of incorporating insecticide resistance monitoring within the Integrated Vector Management initiative.
Assuntos
Aedes , Dengue , Piretrinas , Animais , Feminino , Adulto , Humanos , Aedes/genética , Argentina , Estudos Longitudinais , Mosquitos Vetores/genética , Piretrinas/farmacologia , Dengue/prevenção & controleRESUMO
BACKGROUND: The global impact of Zika virus in Latin America has drawn renewed attention to circulating mosquito-borne viruses in this region, such as dengue and chikungunya. Our objective was to assess socio-ecological factors associated with Aedes mosquito vector density as a measure of arbovirus transmission risk in three cities of potentially recent Zika virus introduction: Ibagué, Colombia; Manta, Ecuador; and Posadas, Argentina, in order to inform disease mitigation strategies. METHODS: We sampled Aedes mosquito populations in a total of 1086 households, using indoor and peridomestic mosquito collection methods, including light traps, resting traps, traps equipped with chemical attractant and aspirators. For each sampled household, we collected socio-economic data using structured questionnaires and data on microenvironmental conditions using iButton data loggers. RESULTS: A total of 3230 female Aedes mosquitoes were collected, of which 99.8% were Aedes aegypti and 0.2% were Aedes albopictus. Mean female Aedes mosquito density per household was 1.71 (standard deviation: 2.84). We used mixed-effects generalized linear Poisson regression analyses to identify predictors of Aedes density, using month, neighborhood and country as random-effects variables. Across study sites, the number of household occupants [incidence rate ratio (IRR): 1.08, 95% confidence interval (CI): 1.01-1.14], presence of entry points for mosquitoes into the household (IRR: 1.51, 95% CI: 1.30-1.76) and presence of decorative vegetation (IRR: 1.52, 95% CI: 1.22-1.88) were associated with higher Aedes density; while being in the highest wealth tertile of household wealth (IRR: 0.78, 95% CI: 0.66-0.92), knowledge of how arboviruses are transmitted (IRR: 0.94, 95% CI: 0.89-1.00) and regular emptying of water containers by occupants (IRR: 0.79, 95% CI: 0.67-0.92) were associated with lower Aedes density. CONCLUSIONS: Our study addresses the complexities of arbovirus vectors of global significance at the interface between human and mosquito populations. Our results point to several predictors of Aedes mosquito vector density in countries with co-circulation of multiple Aedes-borne viruses, and point to modifiable risk factors that may be useful for disease prevention and control.
Assuntos
Aedes/virologia , Distribuição Animal , Infecções por Arbovirus/transmissão , Arbovírus/patogenicidade , Mosquitos Vetores/virologia , Aedes/fisiologia , Animais , Argentina , Febre de Chikungunya/transmissão , Cidades , Colômbia , Dengue/transmissão , Equador , Feminino , Humanos , Mosquitos Vetores/fisiologia , Fatores de Risco , Infecção por Zika virus/transmissãoRESUMO
CRISPR-Cas12a (also called Cpf1) has been commonly used for genomic editing, based on its ability to generate precise double-stranded DNA (dsDNA) breaks. Recently, it was demonstrated that Cas12a exhibits unspecific ssDNAse activity upon target recognition. This feature allows CRISPR-Cas to be coupled with a ssDNA reporter and generate a fast, accurate and ultrasensitive molecular detection method. Here, we demonstrate that Cas12a was able to detect DNA target sequences corresponding to carbapenemases resistance genes such as KPC, NDM and OXA. Also, with the addition of a reverse-transcription step, we were able to detect viral RNA sequences from DENV, ZIKV and HANTV genomes. In all cases, assay run time was less than two hours. Additionally, we report attomolar levels of detection. This methodology was validated using clinical samples from patients infected with Dengue virus. Reactions were visualized by detection of a fluorescent signal, as well as by the use of a simple lateral flow strip. These results indicate that Cas12a is able to detect both DNA and RNA targets, making it an appropriate and convenient tool to detect all types of pathogens.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Farmacorresistência Bacteriana/genética , Endodesoxirribonucleases/genética , Edição de Genes/métodos , Vírus de RNA/genética , beta-Lactamases/farmacologia , DNA de Cadeia Simples/genética , Dengue/virologia , Vírus da Dengue/genética , Corantes Fluorescentes , Vírus Hantaan/genética , Humanos , Técnicas de Diagnóstico Molecular , Vírus de RNA/patogenicidade , RNA Viral/genética , Zika virus/genéticaRESUMO
BACKGROUND: The most common infusion in southern Latin-American countries is prepared with dried leaves of Ilex paraguariensis A. St.-Hil., an aboriginal ancestral beverage known for its high polyphenols concentration currently consumed in > 90% of homes in Argentina, in Paraguay and Uruguay. The economy of entire provinces heavily relies on the production, collection and manufacture of Ilex paraguariensis, the fifth plant species with highest antioxidant activity. Polyphenols are associated to relevant health benefits including strong antioxidant properties. Despite its regional relevance and potential biotechnological applications, little is known about functional genomics and genetics underlying phenotypic variation of relevant traits. By generating tissue specific transcriptomic profiles, we aimed to comprehensively annotate genes in the Ilex paraguariensis phenylpropanoid pathway and to evaluate differential expression profiles. RESULTS: In this study we generated a reliable transcriptome assembly based on a collection of 15 RNA-Seq libraries from different tissues of Ilex paraguariensis. A total of 554 million RNA-Seq reads were assembled into 193,897 transcripts, where 24,612 annotated full-length transcripts had complete ORF. We assessed the transcriptome assembly quality, completeness and accuracy using BUSCO and TransRate; consistency was also evaluated by experimentally validating 11 predicted genes by PCR and sequencing. Functional annotation against KEGG Pathway database identified 1395 unigenes involved in biosynthesis of secondary metabolites, 531 annotated transcripts corresponded to the phenylpropanoid pathway. The top 30 differentially expressed genes among tissue revealed genes involved in photosynthesis and stress response. These significant differences were then validated by qRT-PCR. CONCLUSIONS: Our study is the first to provide data from whole genome gene expression profiles in different Ilex paraguariensis tissues, experimentally validating in-silico predicted genes key to the phenylpropanoid (antioxidant) pathway. Our results provide essential genomic data of potential use in breeding programs for polyphenol content. Further studies are necessary to assess if the observed expression variation in the phenylpropanoid pathway annotated genes is related to variations in leaves' polyphenol content at the population scale. These results set the current reference for Ilex paraguariensis genomic studies and provide a substantial contribution to research and biotechnological applications of phenylpropanoid secondary metabolites.
Assuntos
Genoma de Planta , Ilex paraguariensis/genética , Especificidade de Órgãos/genética , Análise de Sequência de RNA/métodos , Transcriptoma/genética , Regulação da Expressão Gênica de Plantas , Ontologia Genética , Genes de Plantas , Anotação de Sequência Molecular , Folhas de Planta/genética , Raízes de Plantas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Metabolismo Secundário/genéticaRESUMO
BACKGROUND: Bovine leukemia virus (BLV) infection is omnipresent in dairy herds causing direct economic losses due to trade restrictions and lymphosarcoma-related deaths. Milk production drops and increase in the culling rate are also relevant and usually neglected. The BLV provirus persists throughout a lifetime and an inter-individual variation is observed in the level of infection (LI) in vivo. High LI is strongly correlated with disease progression and BLV transmission among herd mates. In a context of high prevalence, classical control strategies are economically prohibitive. Alternatively, host genomics studies aiming to dissect loci associated with LI are potentially useful tools for genetic selection programs tending to abrogate the viral spreading. The LI was measured through the proviral load (PVL) set-point and white blood cells (WBC) counts. The goals of this work were to gain insight into the contribution of SNPs (bovine 50KSNP panel) on LI variability and to identify genomics regions underlying this trait. RESULTS: We quantified anti-p24 response and total leukocytes count in peripheral blood from 1800 cows and used these to select 800 individuals with extreme phenotypes in WBCs and PVL. Two case-control genomic association studies using linear mixed models (LMMs) considering population stratification were performed. The proportion of the variance captured by all QC-passed SNPs represented 0.63 (SE ± 0.14) of the phenotypic variance for PVL and 0.56 (SE ± 0.15) for WBCs. Overall, significant associations (Bonferroni's corrected -log10p > 5.94) were shared for both phenotypes by 24 SNPs within the Bovine MHC. Founder haplotypes were used to measure the linkage disequilibrium (LD) extent (r2 = 0.22 ± 0.27 at inter-SNP distance of 25-50 kb). The SNPs and LD blocks indicated genes potentially associated with LI in infected cows: i.e. relevant immune response related genes (DQA1, DRB3, BOLA-A, LTA, LTB, TNF, IER3, GRP111, CRISP1), several genes involved in cell cytoskeletal reorganization (CD2AP, PKHD1, FLOT1, TUBB5) and modelling of the extracellular matrix (TRAM2, TNXB). Host transcription factors (TFs) were also highlighted (TFAP2D; ABT1, GCM1, PRRC2A). CONCLUSIONS: Data obtained represent a step forward to understand the biology of BLV-bovine interaction, and provide genetic information potentially applicable to selective breeding programs.
Assuntos
Doenças dos Bovinos/genética , Leucose Enzoótica Bovina/genética , Genômica/métodos , Polimorfismo de Nucleotídeo Único , Animais , Bovinos , Doenças dos Bovinos/virologia , Leucose Enzoótica Bovina/virologia , Feminino , Haplótipos , Vírus da Leucemia Bovina/fisiologia , Leucócitos/metabolismo , Leucócitos/virologia , Desequilíbrio de Ligação , Provírus/fisiologia , Fatores de Transcrição/genética , Carga ViralRESUMO
We present the molecular characterization of a new virus infecting yerba mate (Ilex paraguariensis St. Hil.) in Argentina. Deep sequencing of diseased yerba mate plants showing chlorotic linear patterns, chlorotic rings, and vein yellowing resulted in the identification of a new virus resembling plant rhabdoviruses in sequence and genome structure. We have determined the complete genome sequence of this virus, which is 12,876 nt long. Seven open reading frames (ORFs) were identified in the antigenomic orientation of the negative-sense, single-stranded viral RNA, in the order 3'-N-P-P3-P4-M-G-L-5'. Phylogenetic analysis suggested that the described virus is a new member of the genus Cytorhabdovirus, which was supported by the observation of rhabdovirus-like particles within the cytoplasm of infected yerba mate cells. The virus has been tentatively named "yerba mate chlorosis-associated virus" (YmCaV). The availability of the YmCaV genome sequence will contribute to assessing the genetic variability of this virus and determining its role in this yerba mate disease.
Assuntos
Genoma Viral , Ilex paraguariensis/virologia , Vírus de Plantas/genética , RNA Viral/genética , Rhabdoviridae/genética , Argentina , Citoplasma/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Ilex paraguariensis/citologia , Fases de Leitura Aberta , Doenças das Plantas/virologia , Vírus de Plantas/isolamento & purificação , Rhabdoviridae/isolamento & purificaçãoRESUMO
Anadenanthera colubrina var. cebil is a discontinuously distributed native tree species in South American subtropical forests. Thirteen quantitative traits and eight nuclear microsatellite loci were examined in individuals from two biogeographic provinces of Argentina to determine the number and composition of genetically distinguishable groups of individuals and explore possible spatial patterns of the phenotypic and genetic variability. Means of reproductive traits were higher in the Yungas than in the Paranaense biogeographic province, whereas five out of eight nonreproductive quantitative traits showed higher mean values in the latter. Variance coefficients were moderate, and there were significant differences between and within provinces. Three clusters were defined based on spatial model for cluster membership for quantitative traits. One cluster grouped the individuals from the Paranaense biogeographic province whereas the individuals from the Yungas biogeographic province grouped regarding its population of origin. Parameters of molecular genetic variability showed higher values in the Yungas than in the Paranaense biogeographic province. Observed heterozygosity was lower than expected heterozygosity in both biogeographic provinces, indicating an excess of homozygosity. The homozygosity test by Watterson and the exact test by Slatkin suggested diversifying selection for locus Ac41.1. Bayesian clustering spatial model for microsatellites loci data were performed for both all loci and for all loci excluding locus Ac41.1. In both analyses two clusters were inferred. Analysis of molecular variance revealed similar results for all genotypes and for all genotypes defined excluding locus Ac41.1. Most of the total variance is attributable to genetic variation within clusters. The presence of homogeneous clusters was detected for both the phenotypic and molecular genetic variability. Two Bayesian clustering analyses were performed according to molecular genetic data, and two clusters were inferred. Individuals were assigned to their provinces of origin. Genetic molecular variation was higher in the populations of the Yungas biogeographic province which translates into highly qualified populations for conservation. Populations from the Paranaense biogeographic province showed the highest mean value of number of seeds per fruit making them valuable as well with regard to the exploitation of management strategies as a means to recover the impacted areas where these populations are located.
Assuntos
Colubrina/genética , Estudos de Associação Genética , Variação Genética , Genética Populacional , Fenótipo , Argentina , Análise por Conglomerados , Loci Gênicos , Geografia , Repetições de Microssatélites , Característica Quantitativa HerdávelRESUMO
The genetic relationships and structure of fourteen goat (Capra hircus) populations were estimated based on genotyping data from 14 goat populations (n = 410 goats) at 13 microsatellite loci. We used analysis of molecular variance (AMOVA), principal component analysis (PCA) and F statistics (F IS, F IT and F ST) to evaluate the genetic diversity (Ho, He and ad) of the goats. Genetic distances between the 14 goat populations were calculated from allelic frequency data for the 13 microsatellite markers. Moderate differentiation was observed for the populations of the undefined breeds (including the Anglo-Nubian-M breed), the naturalized Brazilian breeds (Moxotó, Canindé), the exotic purebred breeds (Alpine, Saanen, Toggenbourg and Anglo-Nubian) and the naturalized Brazilian Graúna group. Our AMOVA showed that a major portion (88.51 percent) of the total genetic variation resulted from differences between individual goats within populations, while between-populations variation accounted for the remaining 11.49 percent of genetic variation. We used a Reynolds genetic distance matrix and PCA to produce a phenogram based on the 14 goat populations and found three clusters, or groups, consisting of the goats belonging to the undefined breed, the naturalized breeds and the exotic purebred breeds. The closer proximity of the Canindé breed from the Brazilian state of Paraíba to the Graúna breed from the same state than to the genetically conserved Canindé breed from the Brazilian state of Ceará, as well as the heterozygosity values and significant deviations from Hardy-Weinberg equilibrium suggests that there was a high number of homozygotes in the populations studied, and indicates the importance of the State for the conservation of the local breeds. Cataloguing the genetic profile of Brazilian goat populations provides essential information for conservation and genetic improvements programs.