Alvaro Diaz-Badillo

The four serotypes of dengue recognize the same putative receptors in Aedes aegypti midgut and Ae. albopictus cells

BackgroundDengue viruses (DENV) attach to the host cell surface and subsequently enter the cell by receptor-mediated endocytosis. Several primary and low affinity co-receptors for this flavivirus have been identified. However, the presence of these binding molecules on the cell surface does not necessarily render the cell susceptible to infection. Determination of which of them serve as bona fide receptors for this virus in the vector may be relevant to treating DENV infection and in designing control strategies.Results(1) Overlay protein binding assay showed two proteins with molecular masses of 80 and 67 kDa (R80 and R67). (2) Specific antibodies against these two proteins inhibited cell binding and infection. (3) Both proteins were bound by all four serotypes of dengue virus. (4) R80 and R67 were purified by affinity chromatography from Ae. aegypti mosquito midguts and from Ae albopictus C6/36 cells. (5) In addition, a protein with molecular mass of 57 kDa was purified by affinity chromatography from the midgut extracts. (6) R80 and R67 from radiolabeled surface membrane proteins of C6/36 cells were immunoprecipitated by antibodies against Ae. aegypti midgut.ConclusionOur results strongly suggest that R67 and R80 are receptors for the four serotypes of dengue virus in the midgut cells of Ae. aegypti and in C6/36 Ae. albopictus cells.

Multiple recombinants in two dengue virus, serotype-2 isolates from patients from Oaxaca, Mexico

BackgroundDengue (DEN) is a serious cause of mortality and morbidity in the world including Mexico, where the infection is endemic. One of the states with the highest rate of dengue cases is Oaxaca. The cause of DEN is a positive-sense RNA virus, the dengue virus (DENV) that evolves rapidly increasing its variability due to the absence of a repair mechanism that leads to approximately one mutational event per genome replication; which results in enhancement of viral adaptation, including the escape from host immune responses. Additionally, recombination may play a role in driving the evolution of DENV, which may potentially affect virulence and cause host tropism changes. Recombination in DENV has not been described in Mexican strains, neither has been described the relevance in virus evolution in an endemic state such as Oaxaca where the four serotypes of DENV are circulating.ResultsTo study whether there are isolates from Oaxaca having recombination, we obtained the sequence of 6 different isolates of DENV-2 Asian/American genotype from the outbreak 2005-6, one clone of the C(91)-prM-E-NS1(2400) structural genes, and 10 clones of the E gene from the isolate MEX_OAX_1656_05. Evidence of recombination was found by using different methods along with two softwares: RDP3 and GARD. The Oaxaca MEX_OAX_1656_05 and MEX_OAX_1038_05 isolates sequenced in this study were recombinant viruses that incorporate the genome sequence from the Cosmopolitan genotype. Furthermore, the clone of the E gene namely MEX_OAX_165607_05 from this study was also recombinant, incorporating genome sequence from the American genotype.ConclusionsThis is the first report of recombination in DENV-2 in Mexico. Given such a recombinant activity new genomic combinations were produced, this could play a significant role in the DENV evolution and must be considered as a potentially important mechanism generating genetic variation in this virus with serious implications for the vaccines and drugs formulation as occurs for other viruses like poliovirus, influenza and HIV.

Proteomic Identification of Dengue Virus Binding Proteins in Aedes aegypti Mosquitoes and Aedes albopictus Cells

The main vector of dengue in America is the mosquito Aedes aegypti, which is infected by dengue virus (DENV) through receptors of midgut epithelial cells. The envelope protein (E) of dengue virus binds to receptors present on the host cells through its domain III that has been primarily recognized to bind cell receptors. In order to identify potential receptors, proteins from mosquito midgut tissue and C6/36 cells were purified by affinity using columns with the recombinant E protein domain III (rE-DIII) or DENV particles bound covalently to Sepharose 4B to compare and evaluate their performance to bind proteins including putative receptors from female mosquitoes of Ae. aegypti. To determine their identity mass spectrometric analysis of purified proteins separated by polyacrylamide gel electrophoresis was performed. Our results indicate that both viral particles and rE-DIII bound proteins with the same apparent molecular weights of 57 and 67 kDa. In addition, viral particles bound high molecular weight proteins. Purified proteins identified were enolase, beta-adrenergic receptor kinase (beta-ARK), translation elongation factor EF-1 alpha/Tu, and cadherin.

Dengue 2 genotypes in the state of Oaxaca, Mexico

Summary.To genetically characterize dengue 2 (DEN-2) viruses in Oaxaca, Mexico, the C protein, and a portion of the prM protein genes of 8 isolates from the 2001 DEN epidemic were sequenced. The sequences were compared to those of prototype DEN-2 viruses from various parts of the world. Phylogenetic analysis suggested that the 2001 isolates of DEN-2 were of the American/Asian genotype and were most similar to the Jamaica and Venezuelan isolates MARA3, LARD1996 and LARD1910. Molecular analyses confirmed the origin of the isolates. This study indicates that DEN-2 strains of American/Asian genotype probably from Southeast Asian are circulating in Oaxaca.

A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes

Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples.

Extraction and Electrophoresis of DNA from the Remains of Mexican Ancient Populations

Ten years ago, the first reports of human genome sequencing were published in Nature and Science (Venter et al., 2001; Sachidanandam et al., 2001; Lander, 2011). This was very exciting and expectations for the application of genome sequencing technology were high. In the past decade, the cost of sequencing has gone down several orders of magnitude, making it a more accessible technology for research studies. The medical value of comprehensive genome sequencing is now becoming apparent: for example, the genetic cause of a rare and debilitating vascular disorder was solved by genome sequencing at NIH (Jasny and Zahn, 2011; Lander, 2011). It is also possible to solve the genetics of individual Mendelian disorders thereby relating phenotype to genotype. In addition, better treatments for diseases such as cancer, metabolic disorders, inflammation, neurodegeneration or diabetes are expected to be found through studies involving genome sequencing (Lander, 2011). Sequencing also has been used to query variation in populations worldwide, and sequences are now available from extinct hominids as well as from thousands of other species (Rasmussen et al., 2010; Krause et al., 2010; Reich et al., 2010; Balter, 2010; Rasmussen et al., 2010). We expect to know very soon what variation exists among individuals at almost all sites in the genome. This is a great opportunity for population genetics to reconstruct the entire genealogical and mutational history of humans (Callaway, 2011), to understand the evolutionary and genetic forces that affected every region of the genome, to determine disease mutations present in human populations, to elucidate the genetic bases of cognitive and physiological adaptations, and/or to determine the demographic events that led to the colonisation of the earth.

Gene Flow Pattern Among Aedes aegypti Populations in Mexico

Patterns of gene flow vary greatly among Aedes aegypti populations throughout Mexico. The populations are panmictic along the Pacific coast, isolated by distance in northeast Mexico, and exhibit moderate gene flow across the Yucatan peninsula. Nine Ae. aegypti collections from 6 cities in Oaxaca, Mexico, were taken to examine the local patterns of gene flow. Genetic variation was examined in a 387-bp region of the nicotinamide adenine dinucleotide dehydrogenase subunit 4 mitochondrial gene (ND4) using single-strand conformation polymorphism analysis, and 3 haplotypes were detected. Cluster analysis on the linearized FST genetic distances failed to group collections in geographic proximity. Regression analysis of linear or road distances on linearized F(ST) indicated that proximal collections were as diverse as distant collections across an approximately 800-km range. The geographical distribution of the Mexican mosquito haplotype frequencies was determined for the ND4 sequences from 524 individuals from Oaxaca (this study) and 2,043 individuals from our previous studies. Herein, we report on yet another pattern dominated by genetic drift among 9 Ae. aegypti collections from 6 cities in Oaxaca, Mexico, and compare it to those reported in other regions of Mexico. Molecular analysis of variance showed that there was as much genetic variation among collections 4 km apart as there was among all collections. The numbers of haplotypes and the amount of genetic diversity among the collections from Oaxaca were much lower than detected in previous studies in other regions of Mexico and may reflect the effects of control efforts or adaptations to the altitudinal limits (1,500 m) of the species in Mexico. The geographical distribution of mosquito haplotypes in Mexico is also reported. Furthermore, based on the distribution of the mosquito haplotypes in America, we suggest that mosquito dispersion is very efficient, most likely due to commercial transportation.

Association of common polymorphisms in the VEGFA and SIRT1 genes with type 2 diabetes-related traits in Mexicans

Introduction Genetic variants have been replicated for association with type 2 diabetes mellitus (T2D) and many of them with diabetes-related traits. Because T2D is highly prevalent in Mexico, this study aimed to test the association of CDKN2A/B, PPARGC1A, VEGFA, SIRT1 and UCP2 gene polymorphisms (rs10811661, rs8192678, rs2010963, rs7896005 and rs659366 respectively) with metabolic traits in 415 unrelated Mexican mestizos with T2D under three models of inheritance. Material and methods A total of 415 unrelated Mexican mestizos were genotyped by TaqMan assays. Triglycerides, cholesterol, glucose, high-density lipoprotein cholesterol (HDL-C), insulin and anthropometric measurements were determined and the HOMA-IR was calculated. Association studies were tested by the Kruskal-Wallis test, linear regression, statistical power analysis, Bonferroni correction, paired SNP analysis, and physical interaction by GeneMANIA. Results All polymorphisms were in Hardy-Weinberg equilibrium, and the association by genotype with T2D-related traits displayed nominal significance for rs8192678 with glucose (p = 0.023) and triglycerides (p = 0.013); rs2010963 with diastolic blood pressure (DBP) (p = 0.012) and cholesterol (p = 0.013); rs7896005 with DBP (p = 0.012) and insulin (p = 0.011); and rs659366 with cholesterol (p = 0.034), glucose (p = 0.031) and triglycerides (p = 0.028); and the association of rs2010963 with HDL-C (p = 0.0007) was significant. Linear regression performed with three models of inheritance, adjusted by age + sex + BMI and corrected with Bonferroni, showed a significant association of rs2010963 with HDL-C in an additive model (p = 0.007); and rs7896005 was significantly associated with DBP in the recessive model (p = 0.006). Conclusions Rigorous analysis evidenced the association of VEGFA rs2010963 and SIRT1 rs7896005 with HDL-C and DBP respectively; these traits are known predictors of cardiovascular complications, which increase the risk of cardiovascular diseases in this population.