RESUMO
The hydrophobic cuticle is the first line of defense between aerial portions of plants and the external environment. On maize (Zea mays L.) silks, the cuticular cutin matrix is infused with cuticular waxes, consisting of a homologous series of very long-chain fatty acids (VLCFAs), aldehydes, and hydrocarbons. Together with VLC fatty-acyl-CoAs (VLCFA-CoAs), these metabolites serve as precursors, intermediates, and end-products of the cuticular wax biosynthetic pathway. To deconvolute the potentially confounding impacts of the change in silk microenvironment and silk development on this pathway, we profiled cuticular waxes on the silks of the inbreds B73 and Mo17, and their reciprocal hybrids. Multivariate interrogation of these metabolite abundance data demonstrates that VLCFA-CoAs and total free VLCFAs are positively correlated with the cuticular wax metabolome, and this metabolome is primarily affected by changes in the silk microenvironment and plant genotype. Moreover, the genotype effect on the pathway explains the increased accumulation of cuticular hydrocarbons with a concomitant reduction in cuticular VLCFA accumulation on B73 silks, suggesting that the conversion of VLCFA-CoAs to hydrocarbons is more effective in B73 than Mo17. Statistical modeling of the ratios between cuticular hydrocarbons and cuticular VLCFAs reveals a significant role of precursor chain length in determining this ratio. This study establishes the complexity of the product-precursor relationships within the silk cuticular wax-producing network by dissecting both the impact of genotype and the allocation of VLCFA-CoA precursors to different biological processes and demonstrates that longer chain VLCFA-CoAs are preferentially utilized for hydrocarbon biosynthesis.
Assuntos
Ácidos Graxos , Hidrocarbonetos , Ceras , Zea mays , Zea mays/metabolismo , Zea mays/genética , Ceras/metabolismo , Hidrocarbonetos/metabolismo , Ácidos Graxos/metabolismo , Genótipo , Metaboloma , Epiderme Vegetal/metabolismo , Vias BiossintéticasRESUMO
Uncovering the mechanisms regulating hematopoietic specification not only would overcome current limitations related to hematopoietic stem and progenitor cell (HSPC) transplantation, but also advance cellular immunotherapies. However, generating functional human induced pluripotent stem cell (hiPSC)-derived HSPCs and their derivatives has been elusive, necessitating a better understanding of the developmental mechanisms that trigger HSPC specification. Here, we reveal that early activation of the Nod1-Ripk2-NF-kB inflammatory pathway in endothelial cells (ECs) primes them to switch fate towards definitive hemogenic endothelium, a pre-requisite to specify HSPCs. Our genetic and chemical embryonic models show that HSPCs fail to specify in the absence of Nod1 and its downstream kinase Ripk2 due to a failure on hemogenic endothelial (HE) programming, and that small Rho GTPases coordinate the activation of this pathway. Manipulation of NOD1 in a human system of definitive hematopoietic differentiation indicates functional conservation. This work establishes the RAC1-NOD1-RIPK2-NF-kB axis as a critical intrinsic inductor that primes ECs prior to HE fate switch and HSPC specification. Manipulation of this pathway could help derive a competent HE amenable to specify functional patient specific HSPCs and their derivatives for the treatment of blood disorders.
Assuntos
Hemangioblastos , Células-Tronco Pluripotentes Induzidas , Proteínas Monoméricas de Ligação ao GTP , Humanos , Diferenciação Celular , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , NF-kappa B/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Understanding the microbial genomic contributors to antimicrobial resistance (AMR) is essential for early detection of emerging AMR infections, a pressing global health threat in human and veterinary medicine. Here we used whole genome sequencing and antibiotic susceptibility test data from 980 disease causing Escherichia coli isolated from companion and farm animals to model AMR genotypes and phenotypes for 24 antibiotics. We determined the strength of genotype-to-phenotype relationships for 197 AMR genes with elastic net logistic regression. Model predictors were designed to evaluate different potential modes of AMR genotype translation into resistance phenotypes. Our results show a model that considers the presence of individual AMR genes and total number of AMR genes present from a set of genes known to confer resistance was able to accurately predict isolate resistance on average (mean F1 score = 98.0%, SD = 2.3%, mean accuracy = 98.2%, SD = 2.7%). However, fitted models sometimes varied for antibiotics in the same class and for the same antibiotic across animal hosts, suggesting heterogeneity in the genetic determinants of AMR resistance. We conclude that an interpretable AMR prediction model can be used to accurately predict resistance phenotypes across multiple host species and reveal testable hypotheses about how the mechanism of resistance may vary across antibiotics within the same class and across animal hosts for the same antibiotic.
Assuntos
Antibacterianos , Gado , Animais , Humanos , Antibacterianos/farmacologia , Animais de Estimação , Farmacorresistência Bacteriana/genética , Escherichia coli/genéticaRESUMO
The assay for transposase-accessible chromatin with sequencing (ATAC-seq) is a common assay to identify chromatin accessible regions by using a Tn5 transposase that can access, cut, and ligate adapters to DNA fragments for subsequent amplification and sequencing. These sequenced regions are quantified and tested for enrichment in a process referred to as "peak calling." Most unsupervised peak calling methods are based on simple statistical models and suffer from elevated false positive rates. Newly developed supervised deep learning methods can be successful, but they rely on high quality labeled data for training, which can be difficult to obtain. Moreover, though biological replicates are recognized to be important, there are no established approaches for using replicates in the deep learning tools, and the approaches available for traditional methods either cannot be applied to ATAC-seq, where control samples may be unavailable, or are post hoc and do not capitalize on potentially complex, but reproducible signal in the read enrichment data. Here, we propose a novel peak caller that uses unsupervised contrastive learning to extract shared signals from multiple replicates. Raw coverage data are encoded to obtain low-dimensional embeddings and optimized to minimize a contrastive loss over biological replicates. These embeddings are passed to another contrastive loss for learning and predicting peaks and decoded to denoised data under an autoencoder loss. We compared our replicative contrastive learner (RCL) method with other existing methods on ATAC-seq data, using annotations from ChromHMM genomic labels and transcription factor ChIP-seq as noisy truth. RCL consistently achieved the best performance.
Assuntos
Sequenciamento de Cromatina por Imunoprecipitação , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Cromatina/genética , DNA/genéticaRESUMO
MOTIVATION: Allostery enables changes to the dynamic behavior of a protein at distant positions induced by binding. Here, we present APOP, a new allosteric pocket prediction method, which perturbs the pockets formed in the structure by stiffening pairwise interactions in the elastic network across the pocket, to emulate ligand binding. Ranking the pockets based on the shifts in the global mode frequencies, as well as their mean local hydrophobicities, leads to high prediction success when tested on a dataset of allosteric proteins, composed of both monomers and multimeric assemblages. RESULTS: Out of the 104 test cases, APOP predicts known allosteric pockets for 92 within the top 3 rank out of multiple pockets available in the protein. In addition, we demonstrate that APOP can also find new alternative allosteric pockets in proteins. Particularly interesting findings are the discovery of previously overlooked large pockets located in the centers of many protein biological assemblages; binding of ligands at these sites would likely be particularly effective in changing the protein's global dynamics. AVAILABILITY AND IMPLEMENTATION: APOP is freely available as an open-source code (https://github.com/Ambuj-UF/APOP) and as a web server at https://apop.bb.iastate.edu/.
Assuntos
Proteínas , Software , Proteínas/química , Ligantes , Ligação Proteica , Sítios de Ligação , Conformação Proteica , Sítio AlostéricoRESUMO
MOTIVATION: Amplicon sequencing is widely applied to explore heterogeneity and rare variants in genetic populations. Resolving true biological variants and quantifying their abundance is crucial for downstream analyses, but measured abundances are distorted by stochasticity and bias in amplification, plus errors during polymerase chain reaction (PCR) and sequencing. One solution attaches unique molecular identifiers (UMIs) to sample sequences before amplification. Counting UMIs instead of sequences provides unbiased estimates of abundance. While modern methods improve over naïve counting by UMI identity, most do not account for UMI reuse or collision, and they do not adequately model PCR and sequencing errors in the UMIs and sample sequences. RESULTS: We introduce Deduplication and Abundance estimation with UMIs (DAUMI), a probabilistic framework to detect true biological amplicon sequences and accurately estimate their deduplicated abundance. DAUMI recognizes UMI collision, even on highly similar sequences, and detects and corrects most PCR and sequencing errors in the UMI and sampled sequences. DAUMI performs better on simulated and real data compared to other UMI-aware clustering methods. AVAILABILITY AND IMPLEMENTATION: Source code is available at https://github.com/DormanLab/AmpliCI. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Software , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Reação em Cadeia da Polimerase , Análise por ConglomeradosRESUMO
MOTIVATION: Genotyping by sequencing is a powerful tool for investigating genetic variation in plants, but many economically important plants are allopolyploids, where homoeologous similarity obscures the subgenomic origin of reads and confounds allelic and homoeologous SNPs. Recent polyploid genotyping methods use allelic frequencies, rate of heterozygosity, parental cross or other information to resolve read assignment, but good subgenomic references offer the most direct information. The typical strategy aligns reads to the joint reference, performs diploid genotyping within each subgenome, and filters the results, but persistent read misassignment results in an excess of false heterozygous calls. RESULTS: We introduce the Comprehensive Allopolyploid Genotyper (CAPG), which formulates an explicit likelihood to weight read alignments against both subgenomic references and genotype individual allopolyploids from whole-genome resequencing data. We demonstrate CAPG in allotetraploids, where it performs better than Genome Analysis Toolkit's HaplotypeCaller applied to reads aligned to the combined subgenomic references. AVAILABILITY AND IMPLEMENTATION: Code and tutorials are available at https://github.com/Kkulkarni1/CAPG.git. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Técnicas de Genotipagem , Software , Genótipo , Técnicas de Genotipagem/métodos , Análise de Sequência de DNA , Heterozigoto , Alelos , Sequenciamento de Nucleotídeos em Larga Escala/métodosRESUMO
SUMMARY: A new dynamic community identifier (DCI) is presented that relies upon protein residue dynamic cross-correlations generated by Gaussian elastic network models to identify those residue clusters exhibiting motions within a protein. A number of examples of communities are shown for diverse proteins, including GPCRs. It is a tool that can immediately simplify and clarify the most essential functional moving parts of any given protein. Proteins usually can be subdivided into groups of residues that move as communities. These are usually densely packed local sub-structures, but in some cases can be physically distant residues identified to be within the same community. The set of these communities for each protein are the moving parts. The ways in which these are organized overall can aid in understanding many aspects of functional dynamics and allostery. DCI enables a more direct understanding of functions including enzyme activity, action across membranes and changes in the community structure from mutations or ligand binding. The DCI server is freely available on a web site (https://dci.bb.iastate.edu/). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Proteínas de Grãos , Movimento (Física) , Distribuição Normal , Conformação Proteica , Proteínas/químicaRESUMO
BACKGROUND: Angiotensin-converting enzyme inhibitors (ACEIs) are commonly prescribed in dogs, but the ideal dosage is unknown. HYPOTHESIS/OBJECTIVES: In dogs with cardiac disease, a dose-response relationship exists for ACEIs with respect to long-term outcome. ANIMALS: One hundred forty-four dogs with cardiac disease, 63 with current or prior congestive heart failure. METHODS: Retrospective medical record review. Cox proportional hazards models were used to determine variables associated with 2-year survival or survival from first-onset congestive heart failure (CHF). RESULTS: Median initial ACEI dosage was 0.84 (interquartile range [IQR], 0.56-0.98) mg/kg/day, and 108/144 (75%) of dogs received q12h dosing. No clinically relevant changes in renal function test results, serum electrolyte concentrations, or blood pressure occurred between initial prescription of ACEI and first reevaluation (median, 14 days later). In univariable analysis, higher ACEI dose was associated with increased survival from first-onset CHF (P = .005), and within the subgroup of dogs in CHF at the time of ACEI prescription, higher ACEI dose was associated with improved survival at 2 years (P = .04). In multivariable analysis, q12h dose frequency of ACEI (hazard ratio [HR], 0.30; 95% CI, 0.10-0.88; P = .03) and higher serum potassium concentration at visit 1 (HR, 0.39; 95% CI, 0.16-0.97; P = .04) were predictive of 2-year survival. The ACEIs were well-tolerated, with only 8/144 (5.6%) dogs having ACEI dose decreased or discontinued because of adverse effects. CONCLUSIONS AND CLINICAL IMPORTANCE: Twice daily dose frequency might optimize the cardioprotective benefit of ACEIs.
Assuntos
Doenças do Cão , Insuficiência Cardíaca , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Animais , Doenças do Cão/tratamento farmacológico , Cães , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/veterinária , Potássio , Modelos de Riscos Proporcionais , Estudos RetrospectivosRESUMO
Protein-protein interaction networks are one of the most effective representations of cellular behavior. In order to build these models, high-throughput techniques are required. Next-generation interaction screening (NGIS) protocols that combine yeast two-hybrid (Y2H) with deep sequencing are promising approaches to generate interactome networks in any organism. However, challenges remain to mining reliable information from these screens and thus, limit its broader implementation. Here, we present a computational framework, designated Y2H-SCORES, for analyzing high-throughput Y2H screens. Y2H-SCORES considers key aspects of NGIS experimental design and important characteristics of the resulting data that distinguish it from RNA-seq expression datasets. Three quantitative ranking scores were implemented to identify interacting partners, comprising: 1) significant enrichment under selection for positive interactions, 2) degree of interaction specificity among multi-bait comparisons, and 3) selection of in-frame interactors. Using simulation and an empirical dataset, we provide a quantitative assessment to predict interacting partners under a wide range of experimental scenarios, facilitating independent confirmation by one-to-one bait-prey tests. Simulation of Y2H-NGIS enabled us to identify conditions that maximize detection of true interactors, which can be achieved with protocols such as prey library normalization, maintenance of larger culture volumes and replication of experimental treatments. Y2H-SCORES can be implemented in different yeast-based interaction screenings, with an equivalent or superior performance than existing methods. Proof-of-concept was demonstrated by discovery and validation of novel interactions between the barley nucleotide-binding leucine-rich repeat (NLR) immune receptor MLA6, and fourteen proteins, including those that function in signaling, transcriptional regulation, and intracellular trafficking.
Assuntos
Proteínas de Plantas/metabolismo , Mapas de Interação de Proteínas , Receptores Imunológicos/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Conjuntos de Dados como Assunto , Estudo de Prova de ConceitoRESUMO
MOTIVATION: Next-generation amplicon sequencing is a powerful tool for investigating microbial communities. A main challenge is to distinguish true biological variants from errors caused by amplification and sequencing. In traditional analyses, such errors are eliminated by clustering reads within a sequence similarity threshold, usually 97%, and constructing operational taxonomic units, but the arbitrary threshold leads to low resolution and high false-positive rates. Recently developed 'denoising' methods have proven able to resolve single-nucleotide amplicon variants, but they still miss low-frequency sequences, especially those near more frequent sequences, because they ignore the sequencing quality information. RESULTS: We introduce AmpliCI, a reference-free, model-based method for rapidly resolving the number, abundance and identity of error-free sequences in massive Illumina amplicon datasets. AmpliCI considers the quality information and allows the data, not an arbitrary threshold or an external database, to drive conclusions. AmpliCI estimates a finite mixture model, using a greedy strategy to gradually select error-free sequences and approximately maximize the likelihood. AmpliCI has better performance than three popular denoising methods, with acceptable computation time and memory usage. AVAILABILITY AND IMPLEMENTATION: Source code is available at https://github.com/DormanLab/AmpliCI. SUPPLEMENTARY INFORMATION: Supplementary material are available at Bioinformatics online.
Assuntos
Algoritmos , Microbiota , Análise por Conglomerados , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA , SoftwareRESUMO
Post-weaning diarrhea caused by enterotoxigenic E. coli (ETEC) causes significant economic losses for pig producers. This study was to test the hypotheses that an ETEC challenge disrupts intestinal microbial homeostasis and the inclusion of dietary soluble (10% sugar beet pulp) or insoluble fiber (15% corn distillers dried grains with solubles) with or without exogenous carbohydrases will protect or restore the gut microbial homeostasis in weaned pigs. Sixty crossbred piglets (6.9 ± 0.1 kg) were blocked by body weight and randomly assigned to one of six treatments (n = 10), including a non-challenged control (NC), ETEC F18-challenged positive control (PC), ETEC-challenged soluble fiber without (SF-) or with carbohydrases (SF+), and ETEC-challenged insoluble fiber without (IF-) or with carbohydrases (IF+). Pigs were housed individually and orally received either ETEC inoculum or PBS-sham inoculum on day 7 post-weaning. Intestinal contents were collected on day 14 or 15. The V4 region of the bacterial 16S rRNA was amplified and sequenced. High-quality reads (total 6,671,739) were selected and clustered into 3,330 OTUs. No differences were observed in α-diversity among treatments. The ileal microbiota in NC and PC had modest separation in the weighted PCoA plot; the microbial structures were slightly altered by SF+ and IF- compared with PC. The PC increased ileal Escherichia-Shigella (P < 0.01) and numerically decreased Lactobacillus compared to NC. Predicted functional pathways enriched in the ileal microbiota of PC pigs indicated enhanced activity of Gram-negative bacteria, in agreement with increased Escherichia-Shigella. The SF+ tended to decrease (P < 0.10) ileal Escherichia-Shigella compared to PC. Greater abundance of ileal Streptococcus, Turicibacter, and Roseburia and colonic Prevotella were observed in SF- and SF+ than PC (P < 0.05). Pigs fed IF + had greater Lactobacillus and Roseburia than PC pigs (P < 0.05). The ETEC challenge reduced total volatile fatty acid (VFA) compared with NC (P < 0.05). The SF+ tended to increase (P < 0.10) and SF- significantly increased (P < 0.05) colonic total VFA compared with PC. Collectively, ETEC challenge disrupted gut microbial homeostasis and impaired microbial fermentation capacity. Soluble fiber improved VFA production. Dietary fiber and carbohydrases altered microbiota composition to maintain or restore microbial homeostasis.
RESUMO
Understanding the dynamics of acute viral infection is crucial for developing strategies to prevent and control infection. In this study, lentiviral dynamics in a host without adaptive immunity were examined in order to determine kinetic parameters of infection and quantify the effect of neutralizing antibodies in preventing infection, using mathematical modeling of data from equine infectious anemia virus (EIAV) infection of horses with severe combined immunodeficiency (SCID). Estimated parameters were used to calculate the basic reproductive number and virus doubling time and found that the rate that antibodies neutralized virus was ~18 times greater than the virus clearance rate. These results establish EIAV replication kinetics in SCID horses and the minimal efficacy of antibodies that blocked infection. Furthermore, they indicate that EIAV is at most mildly cytopathic. This study advances our understanding of EIAV infection and may have important implications for the control of other viral infections, including HIV.
Assuntos
Anemia Infecciosa Equina/prevenção & controle , Anemia Infecciosa Equina/virologia , Vírus da Anemia Infecciosa Equina/imunologia , Vírus da Anemia Infecciosa Equina/isolamento & purificação , Carga Viral , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Cavalos , Modelos Teóricos , Imunodeficiência Combinada Severa/complicações , Imunodeficiência Combinada Severa/veterináriaRESUMO
Transmission of influenza A virus (IAV) from humans to swine occurs with relative frequency and is a critical contributor to swine IAV diversity. Subsequent to the introduction of these human seasonal lineages, there is often reassortment with endemic viruses and antigenic drift. To address whether particular genome constellations contributed to viral persistence following the introduction of the 2009 H1N1 human pandemic virus to swine in the USA, we collated and analysed 616 whole genomes of swine H1 isolates. For each gene, sequences were aligned, the best-known maximum likelihood phylogeny was inferred, and each virus was assigned a clade based upon its evolutionary history. A time-scaled Bayesian approach was implemented for the haemagglutinin (HA) gene to determine the patterns of genetic diversity over time. From these analyses, we observed an increase in genome diversity across all H1 lineages and clades, with the H1-γ and H1-δ1 genetic clades containing the greatest number of unique genome patterns. We documented 74 genome patterns from 2009 to 2016, of which 3 genome patterns were consistently detected at a significantly higher level than others across the entire time period. Eight genome patterns increased significantly, while five genome patterns were shown to decline in detection over time. Viruses with genome patterns identified as persisting in the US swine population may possess a greater capacity to infect and transmit in swine. This study highlights the emerging genetic diversity of US swine IAV from 2009 to 2016, with implications for swine and public health and vaccine control efforts.
Assuntos
Genoma Viral , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Infecções por Orthomyxoviridae/veterinária , Doenças dos Suínos/virologia , Animais , Evolução Molecular , Genômica , Genótipo , Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H1N1/genética , Infecções por Orthomyxoviridae/virologia , Filogenia , RNA Viral/genética , Suínos , Estados UnidosRESUMO
BACKGROUND: Rev-like proteins are post-transcriptional regulatory proteins found in several retrovirus genera, including lentiviruses, betaretroviruses, and deltaretroviruses. These essential proteins mediate the nuclear export of incompletely spliced viral RNA, and act by tethering viral pre-mRNA to the host CRM1 nuclear export machinery. Although all Rev-like proteins are functionally homologous, they share less than 30% sequence identity. In the present study, we computationally assessed the extent of structural homology among retroviral Rev-like proteins within a phylogenetic framework. RESULTS: We undertook a comprehensive analysis of overall protein domain architecture and predicted secondary structural features for representative members of the Rev-like family of proteins. Similar patterns of α-helical domains were identified for Rev-like proteins within each genus, with the exception of deltaretroviruses, which were devoid of α-helices. Coiled-coil oligomerization motifs were also identified for most Rev-like proteins, with the notable exceptions of HIV-1, the deltaretroviruses, and some small ruminant lentiviruses. In Rev proteins of primate lentiviruses, the presence of predicted coiled-coil motifs segregated within specific primate lineages: HIV-1 descended from SIVs that lacked predicted coiled-coils in Rev whereas HIV-2 descended from SIVs that contained predicted coiled-coils in Rev. Phylogenetic ancestral reconstruction of coiled-coils for all Rev-like proteins predicted a single origin for the coiled-coil motif, followed by three losses of the predicted signal. The absence of a coiled-coil signal in HIV-1 was associated with replacement of canonical polar residues with non-canonical hydrophobic residues. However, hydrophobic residues were retained in the key 'a' and 'd' positions, and the α-helical region of HIV-1 Rev oligomerization domain could be modeled as a helical wheel with two predicted interaction interfaces. Moreover, the predicted interfaces mapped to the dimerization and oligomerization interfaces in HIV-1 Rev crystal structures. Helical wheel projections of other retroviral Rev-like proteins, including endogenous sequences, revealed similar interaction interfaces that could mediate oligomerization. CONCLUSIONS: Sequence-based computational analyses of Rev-like proteins, together with helical wheel projections of oligomerization domains, reveal a conserved homogeneous structural basis for oligomerization by retroviral Rev-like proteins.
Assuntos
Produtos do Gene rev/química , Produtos do Gene rev/metabolismo , Modelos Moleculares , Retroviridae/química , Retroviridae/metabolismo , Sequência de Aminoácidos , Dimerização , Variação Genética , Filogenia , Estrutura Secundária de Proteína , Proteínas dos Retroviridae/química , Proteínas dos Retroviridae/metabolismo , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: We previously reported the discovery of a novel, putative flavivirus designated T'Ho virus in Culex quinquefasciatus mosquitoes in the Yucatan Peninsula of Mexico. A 1358-nt region of the NS5 gene was amplified and sequenced but an isolate was not recovered. RESULTS: The complete genome of T'Ho virus was sequenced using a combination of unbiased high-throughput sequencing, 5' and 3' rapid amplification of cDNA ends, reverse transcription-polymerase chain reaction and Sanger sequencing. The genome contains a single open reading frame of 10,284 nt which is flanked by 5' and 3' untranslated regions of 97 and 556-nt, respectively. Genome sequence alignments revealed that T'Ho virus is most closely related to Rocio virus (67.4% nucleotide identity) and Ilheus virus (65.9%), both of which belong to the Ntaya group, followed by other Ntaya group viruses (58.8-63.3%) and Japanese encephalitis group viruses (62.0-63.7%). Phylogenetic inference is in agreement with these findings. CONCLUSIONS: This study furthers our understanding of flavivirus genetics, phylogeny and diagnostics. Because the two closest known relatives of T'Ho virus are human pathogens, T'Ho virus could be an unrecognized cause of human disease. It is therefore important that future studies investigate the public health significance of this virus.
Assuntos
Flavivirus/genética , Análise de Sequência de DNA , Sequenciamento Completo do Genoma , Animais , Análise por Conglomerados , Culex , Flavivirus/isolamento & purificação , México , Fases de Leitura Aberta , Filogenia , Homologia de Sequência do Ácido NucleicoRESUMO
BACKGROUND: Baseline hematologic data are used by veterinarians and wildlife biologists to perform health assessments on target animal species. Hematologic measurements are influenced by various factors including geography. Baseline hematologic RIs have been established for domestic water buffaloes (Bubalus bubalis) from various countries in the Eastern Hemisphere, but these data are not readily available for the Western Hemisphere. OBJECTIVE: The aim of this study was to determine hematologic values for domestic water buffaloes from several commercial farms in southern Mexico. METHODS: Blood was collected from 126 healthy, postlactating females (3 to 10 years old) from the Murrah breed, and 10 hematologic variables were measured. RESULTS: Means, SDs, RIs, medians (MED), median absolute deviations (MAD), and other statistics were calculated for each hematologic variable. The MED (and MAD) for each variable are as follows: RBC count, 7.6 (1.1) × 1012 /L; hemoglobin, 116.0 (13.3) g/L; PCV, 41.5 (7.6) %; MCV, 56.8 (7.0) fL; MCH, 14.6 (1.6) pg; MCHC, 250.0 (35.6) g/L; RDW (SD), 29.7 (5.5) fL; RDW (CV), 18.2 (1.4) %; reticulocytes, 0.0 (0.0) %, and WBC count, 12.4 (1.3) × 109 /L. These values were compared to those previously reported for water buffaloes from several countries in the Eastern Hemisphere and, on most occasions, they differed significantly. CONCLUSIONS: Our data can be used by veterinarians and other personnel involved in buffalo production in Mexico during medical evaluations.
Assuntos
Búfalos/sangue , Animais , Contagem de Eritrócitos/veterinária , Índices de Eritrócitos/veterinária , Feminino , Hematócrito/veterinária , Hemoglobinas/análise , Contagem de Leucócitos/veterinária , México , Valores de ReferênciaRESUMO
Genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) challenges efforts to develop effective and broadly acting vaccines. Although genetic variation in PRRSV has been extensively documented, the effects of this variation on virus phenotype are less well understood. In the present study, PRRSV open reading frame (ORF)2-6 variants predominant during the first six weeks following experimental infection were characterized for antigenic and replication phenotype. There was limited genetic variation during these early times after infection; however, distinct ORF2-6 haplotypes that differed from the NVSL97-7895 inoculum were identified in each of the five pigs examined. Chimeric viruses containing all or part of predominant ORF2-6 haplotypes were constructed and tested in virus neutralization and in vitro replication assays. In two pigs, genetic variation in ORF2-6 resulted in increased resistance to neutralization by autologous sera. Mapping studies indicated that variation in either ORF2-4 or ORF5-6 could confer increased neutralization resistance, but there was no single amino acid substitution that was predictive of neutralization phenotype. Detailed analyses of the early steps in PRRSV replication in the presence and absence of neutralizing antibody revealed both significant inhibition of virion attachment and, independently, a significant delay in the appearance of newly synthesized viral RNA. In all pigs, genetic variation in ORF2-6 also resulted in significant reduction in infectivity on MARC-145 cells, suggesting variation in ORF2-6 may also be important for virus replication in vivo. Together, these data reveal that variation appearing early after infection, though limited, alters important virus phenotypes and contributes to antigenic and biologic diversity of PRRSV.
Assuntos
Variação Antigênica/genética , Variação Antigênica/imunologia , Variação Genética , Fases de Leitura Aberta/genética , Fases de Leitura Aberta/imunologia , Síndrome Respiratória e Reprodutiva Suína/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes , Antígenos Virais/genética , Antígenos Virais/imunologia , Sequência de Bases , Linhagem Celular , Modelos Animais de Doenças , Fenótipo , Síndrome Respiratória e Reprodutiva Suína/sangue , RNA Viral/genética , Sus scrofa , Suínos , Vírion , Ligação Viral , Replicação ViralRESUMO
Nucleotide sequencing was performed on part of the medium and large genome segments of 17 Cache Valley virus (CVV) isolates from the Yucatan Peninsula of Mexico. Alignment of these sequences to all other sequences in the Genbank database revealed that they have greatest nucleotide identity (97-98 %) with the equivalent regions of Tlacotalpan virus (TLAV), which is considered to be a variety of CVV. Next, cross-plaque reduction neutralization tests (PRNTs) were performed using sera from mice that had been inoculated with a representative isolate from the Yucatan Peninsula (CVV-478) or the prototype TLAV isolate (61-D-240). The PRNT titers exhibited a twofold difference in one direction and no difference in the other direction suggesting that CVV-478 and 61-D-240 belong to the same CVV subtype. In conclusion, we demonstrate that the CVV isolates from the Yucatan Peninsula of Mexico are genetically and antigenically similar to the prototype TLAV isolate.
Assuntos
Aedes/virologia , Vírus Bunyamwera/genética , Vírus Bunyamwera/imunologia , Animais , Vírus Bunyamwera/classificação , Vírus Bunyamwera/isolamento & purificação , Feminino , Soros Imunes/imunologia , México , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Filogenia , Análise de Sequência de DNA , Ensaio de Placa ViralRESUMO
We previously reported the isolation of South River virus (SORV) from a pool of mosquitoes collected in the Yucatan Peninsula of Mexico (Farfan-Ale et al. in Vector Borne Zoonotic Dis 10:777-783, 5). The isolate (designated SORV-252) was identified as SORV after a 197-nucleotide region of its small RNA genome segment was sequenced. In the present study, the complete small and medium RNA genome segments and part of the large RNA genome segment of SORV-252 were sequenced and shown to have 92%, 85% and 90% nucleotide sequence identity, respectively, to the homologous regions of the prototype SORV isolate (NJO-94F). To determine the antigenic relationship between SORV-252 and NJO-94F, cross-plaque reduction neutralization tests (PRNTs) were performed using sera from mice inoculated with these viruses. SORV-252 and NJO-94F were distinguishable in the cross-neutralization assays; there was a twofold difference in the PRNT titers in one direction and a fourfold difference in the other direction, suggesting that SORV-252 represents a novel subtype of SORV. Additionally, SORV-252 and NJO-94F have distinct plaque morphologies in African green monkey kidney (Vero) cells. In conclusion, we provide evidence that a novel subtype of SORV is present in the Yucatan Peninsula of Mexico.