Computational protocol: Re-emergence of dengue virus serotype 2 strains in the 2013 outbreak in Nepal

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Protocol publication

[…] Study design and locations: A hospital-based, prospective study was carried out from July to December 2013. Six hospitals in the outbreak regions- Narayani zone hospital and Bhawani Hospital of Birgunj, Parsa; Bharatpur Hospital in Bharatpur, Chitwan; Universal Medical College in Bhairahawa; Institute of Medicine in Kathmandu; and Janakpur Zone Hospital in Janakpur, Dhanusa, were identified as the centres for collection of biological samples. Selected samples were brought to the laboratory in the Central Department of Biotechnology, Tribhuvan University, Kathmandu in Nepal for serological and reverse transcription (RT)-PCR analysis and cDNAs were sent to the collaborator's laboratory in India for nucleotide sequencing and phylogenetic analysis.Inclusion and exclusion criteria of patients: All the ‘dengue-suspected’ patients who visited the above hospitals were routinely screened for dengue infection using a rapid diagnostic kit (RDT) (Panbio, Australia) supplied by the Ministry of Health and Family Welfare, Government of Nepal for the detection of IgM antibodies. The WHO-2009 definition was used to identify patients suspected to have dengue and these patients were subjected to the rapid diagnostic test. A total of 2340 dengue-suspected patients visited these hospitals during the study period. Consent was obtained from 198 patients (Parsa n=127; Chitwan n=33; Rupandehi n=6; Janakpur n=24 and Kathmandu n=8) based on the available testing facilities in our laboratories. These patients underwent detailed clinical and laboratory investigations.Ethical approval was taken from Nepal Health Research Council (NHRC), Kathmandu to conduct the study using human samples as well as transferring the non-infectious cDNA material to the collaborator's laboratory.Collection of blood samples and serological assay: Blood samples (3 ml) were collected from the 198 patients and the serum was separated and stored in deep freezers at the respective hospitals. The samples were brought to our laboratory in cold-chain and stored further at -80° C. All these samples were further subjected to Dengue IgM capture ELISA (Panbio, Australia) according to the instructions given in the manufacturer's protocol. Panbio units were computed for each sample, and results were classified either as positive or negative.Viral RNA detection and serotype identification: Viral RNA was isolated from 52 samples using Nucleospin viral RNA isolation kit (MACHEREY-NAGEL, Germany) following the manufacturer's instructions. Dengue viral RNA detection was done by RT-PCR using the specific pair of primers D1F and Dencom R2 which amplified the region corresponding to the nucleotide positions 134-785 of the genome coding for the Core-Pre-membrane (C-PrM) region as described previously (). For serotype identification, a semi-nested PCR assay was performed on the RT-PCR positive samples with the 1:10 diluted primary PCR product as the template and previously reported serotype-specific primers (D1F, NTS1, NTS2, NTS3 and NDen4; ) using GoTaq PCR Master mix (Promega, USA).Nucleotide sequencing and phylogenetic analysis: For nucleotide sequencing, the C-prM region was PCR amplified using the D1F-DencomR2 primers from the first strand viral cDNA that was synthesized using AMV RT-reverse transcription system (Promega, USA). The PCR product was purified by Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare, USA). Both strands of the amplicon were directly sequenced using the specific primers D1F and DencomR2. The Big Dye Terminator kit (Applied Biosystems, USA) was used as per the manufacturer's instructions for sequencing and the reaction mix was analyzed in an ABI 3730 Genetic Analyzer automated DNA sequencer. The sequences were aligned using CLUSTAL W function of BioEdit 7.2.3 software package and the phylogenetic tree was constructed with the Maximum-likelihood (ML) method using MEGA 6 (version 6.0) software package (www.megasoftware.net/). […]

Pipeline specifications

Software tools Clustal W, BioEdit, MEGA
Application Phylogenetics
Organisms Homo sapiens
Diseases Dengue, Exanthema, Thrombocytopenia
Chemicals Nucleotides