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[…] Human and arthropod sampling: After receiving approval from the institutional Ethics Committee (CEP/HUJM/100/2011), serum samples and epidemiological data were obtained from 604 patients in 20 cities across MT who sought medical care between October, 2011 and July, 2012 for acute febrile illnesses lasting less than five days. Also, 3,433 female Culex spp. were captured with Nasci aspirators from 184 censitary sectors of Cuiabá between January and May, 2013, identified using GPS locators. Three places were sampled at each sector. These Culicidae were identified according to dichotomy keys , and a nested-PCR for Culex quinquefasciatus and allocated to 409 pools of between one and ten mosquitoes; 403 with 3,425 Cx. quinquefasciatus, five with seven Cx. bidens or Cx. interfor and one with one female of Culex spinosus. Flaviviruses detection: Viral RNA from patient serum and total RNA from mosquito pools were extracted according to manufacturers' instructions (QIAamp Viral RNA Mini Kit, Qiagen and Trizol, Invitrogen, respectively). Extracted RNA was subject to a multiplex semi-nested reverse transcription PCR (RT-PCR) for a nucleotide region of flaviviruses NS5 gene (958 bp), followed by a species-specific secondary reaction differentiating 11 flaviviruses, as previously described. Flavivirus-positive samples were confirmed by at least two independent single reactions with the same forward and species-specific reverse primer. PCR products were then submitted to nucleotide sequencing (3500 Genetic Analyzer, Applied Biossystems, USA). RNA from the SLEV strain genotype V-B BeH 355964 and no template were included as controls in all the reactions. Nucleotide sequences obtained from the positive control were analyzed to exclude contamination.The minimum infection rate (MIR) was calculated with the formula ([number of positive pools / total specimens tested] x 1000), considering the total of Culexquinquefasciatus specimens tested (3,433 mosquitoes). SLEV-positive samples were subjected to inoculation in C6/36 cells. Nucleotide and amino acid sequence analysis of an envelope gene region from SLEV: A region of the SLEV envelope gene (477 bp) was amplified in positive samples via semi-nested RT - PCR and sequenced for phylogenetic analysis. A phylogenetic tree was constructed with the neighbor-joining method, based on the Tamura-Nei distance model and 1,000 bootstrap replicates (Geneious R7 7.1.7, USA) using reference SLEV sequences from the GenBank database (PubMed, NCBI, USA). Deduced amino acid sequences were also analyzed (Geneious R7 version 7.1.7; Molecular Evolutionary Genetics Analysis version 5.05, USA), including residues present at specific positions characteristic of SLEV lineages.Nucleotide sequences obtained in this study were deposited at GenBank, pubMed (accession numbers: KJ699354; KJ957827; KJ847419; KJ801827). […]

Pipeline specifications

Software tools Geneious, MEGA
Applications Phylogenetics, Protein sequence analysis
Organisms Saint Louis encephalitis virus, Homo sapiens, Culex quinquefasciatus
Diseases Dengue, Encephalitis, Encephalitis, Arbovirus, Yellow Fever, Coinfection