Computational protocol: Detection of Oropouche virus segment S in patients and inCulexquinquefasciatus in the state of Mato Grosso, Brazil

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Protocol publication

[…] Clinical samples and ethics statement - In this study, serum samples from 529 patients with acute febrile illness persisting for up to five days from 17 cities of MT were obtained between October 2011-July 2012 in the Public Health Central Laboratory (LACEN-MT). All samples had been tested previously for DENV serotypes and YFV by virus isolation followed by immunofluorescence and molecular techniques.Serum samples were stored at -80°C at the Laboratory of Virology of the School or Medicine of the Federal University of Mato Grosso (UFMT). The viral RNA was extracted (Qiamp viral RNA mini kit; Qiagen, Germany) and immediately converted into genus-specific cDNA. Nested-reverse transcription-polymerase chain reaction (RT-PCR) for the segment S of the Simbu serogroup of the Orthobunyavirus genus was performed ().The procedures involving human samples were previously approved by the institutional review board of the Julio Muller University Hospital Ethical Committee on Research under the register 100/2011. All epidemiological data obtained through the Information System for Notifiable Diseases records and/or directly from the patients were handled anonymously and confidentially. Sampling of Culex mosquitoes in Cuiabá - Because most of the patients included in the study are residents of the metropolitan area of Cuiabá, a parallel study was conducted with mosquitoes. Specimens of Cx. quinquefasciatus (n = 387) were captured between 01:00 pm-05:00 pm during the rainy season (January-April 2013) with Nasci aspirators and hand nets from three places in each of 200 censitary sectors that were randomly selected in Cuiabá. The mosquitoes remained in the laboratory for at least 12 h, receiving artificial feeding with sugar water until identification with a dichotomy key () and with a molecular approach using semi-nested-PCR for Cx. quinquefasciatus (). Pools containing one-35 mosquito specimens according to place of capture, genus, species and sex were stored at -80ºC. Only female pools were included in the present study.Pools were macerated and diluted in RNAse-free phosphate-buffered solution; 400 μL of the supernatant was used for total RNA and total DNA extraction (Trizol; Invitrogen, USA) and cDNA was immediately synthesised with Superscript III (Invitrogen) following the manufacturer’s instructions. The extracted DNA was used for molecular confirmation of the mosquito species. Semi-nested-PCR for Culex species - The protocol used forCx. quinquefasciatus identification was performed in 78 pools that were identified as Culex pipiens complexand 102 of Culex spp according to with few modifications. In the first reaction, the primers B1246s (0.2 µM) and F1475 (0.2 µM) were used with the following cycling conditions: 94ºC for 5 min, 35 cycles of 94ºC for 30 s, 55ºC for 30 s and 72ºC for 1 min and a final extension at 72ºC for 5 min. This PCR product was subjected to semi-nested-PCR using 1 ng of PCR product, the primers B1246s (0.2 µM) and ACEquin (0.8 µM) and cycling conditions as follows: 94ºC for 5 min, 35 cycles of 94ºC for 30 s, 57ºC for 30 s, 72ºC for 1 min and final extension of 72ºC for 5 min. Nested-PCR for segment S of orthobunyaviruses belonging to the Simbu serogroup - The protocol described by was used to amplify segment S from the genome of orthobunyaviruses (961 bp) with a few modifications. Briefly, cDNA (8 µL) was amplified with BUN-S primer and was then subjected to a PCR reaction containing 10x PCR buffer, MgCl2 (2 mM), dNTPs (0.2 mM), the primers BUN-S (+) (0.6 µM) and BUN-C (-) (0.6 µM), 1 U of DNA polymerase (LGC Biotecnologia, Brazil) and ultrapure water for 50 µL of reaction following the cycling conditions described by the authors. The second PCR reaction targets a 300-bp region of Simbu serogroup members (BS-S and BS-C primers). This reaction was performed using 2 µL of the product of the first reaction and the same concentrations of reagents and cycling conditions, with a final volume of 25 µL. cDNA from an OROV strain (BeAn19991) and no template were included as controls; precautions to avoid contamination were undertaken during procedures. The positive control was sequenced to rule out contamination. Nucleotide sequencing and phylogenetic analysis - The sequencing was performed using POP-7TM and an ABI 3130 DNA Sequencer. Approximately 10-40 ng of purified nested-PCR product (300 bp of segment S) was amplified following the BigDye Terminator v.3.1 Cycle Sequencing protocol. The sequences were initially filtered by applying a Phred score cut-off of ≤ 20 using the Sequencing Analysis (Applied Biosystems, v.5.3.1) software, a procedure that was kindly performed by the Leônidas e Maria Deane Institute, Oswaldo Cruz Foundation (Fiocruz) Amazônia. Only the filtered sequences were considered for contig assembly after trimming the low-quality ends. Geneious R6 (Biomatters, v.6.0.5) was used for this purpose. The contigs were compared with reference sequences through the nucleotide Basic Local Alignment Search Tool (BLASTn, GenBank, PubMed).Phylogenetic analysis included several nucleotide sequences of segment S from OROV strains available from GenBank (PubMed, National Center for Biotechnology Information). After alignment with CLUSTALW and analysis using Molecular Evolutionary Genetics Analysis (MEGA v.5.05), the best model of nucleotide substitution was determined by jModelTest (v.2.2.6). A phylogenetic tree was generated using a region of the N protein of OROV with 1,000 bootstrap replicates. The evolutionary history was inferred using the neighbour-joining method with Tamura three-parameter distance model and the rate variation among sites was modelled with a gamma distribution (shape parameter = 1). Outgroups included the Buttonwillow, Faceys Paddock, Ingwavuma, Mermet, Aino, Tinaroo and Akabane orthobunyaviruses. Inoculation in cell culture - Samples positive for OROV according to nested-RT-PCR and nucleotide sequencing were diluted 1:10 and inoculated into 24-well polystyrene plates containing Vero cells (ATCC CCL-81). The cells were cultivated in RPMI-1640 medium supplemented with 5% foetal bovine serum (FBS). After the incubation period (2 h) at 37ºC and 5% CO2, the inoculum was removed and the monolayer was washed with RPMI-1640 medium containing antimycotic agents and antibiotics. The culture medium was replaced and the cells were observed daily for seven-10 days. After this period, the monolayers were harvested for total RNA extraction (Trizol) followed by nested-RT-PCR and sequencing, as previously described. Three passages were performed with the supernatant to ensure viral amplification. Data analysis - The minimum infection rate (MIR) was calculated with the formula (number of positive pools/total specimens tested) x 1,000, considering the total of Cx. quinquefasciatus specimens tested (3,425 mosquitoes). The geospatial data were analysed with ArcMap (ESri ArcGIS, v.9.3). Accessions - Nucleotide sequences from the segment S of OROV obtained in the present study were deposited in GenBank and PubMed with the accessions KP310500-KP310507 (mosquito pools) and KP347671-KP347674; KP954633 (human samples). […]

Pipeline specifications

Software tools Geneious, BLASTN, Clustal W, MEGA, MEGA-V, jModelTest
Applications Phylogenetics, GWAS
Organisms Simbu orthobunyavirus, Homo sapiens, Oropouche virus
Chemicals Nucleotides