Computational protocol: Characterization of Newcastle disease virus isolates obtained from outbreak cases in commercial chickens and wild pigeons in Ethiopia

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Protocol publication

[…] To determine the amino acid sequence of the fusion protein cleavage site, the viral RNA was extracted directly from infected allantoic fluids for each isolate using the Nucleospin RNA II kit (Machery-Nagel, Duren Germany) according to the manufacturer’s instructions. Amplification was performed with primers NOH-For (5′-TAC ACC TCA TCC CAG ACA GG-3′) and NOH-Rev (5′-AGT CGG AGG ATG TGT TGG CAG C-3′) (Terregino and Capua ). The amplified DNA products were checked by electrophoresis through 1 % agarose gel and purified using ExoSAPIT (USB Corporation, Cleveland, OH). DNA products were subjected to direct nucleotide sequencing using v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster city, CA, USA) and sequenced in a 16-capillary ABI PRISM 3130xl Genetic Analyzer (Applied Biosystem).Pairwise sequence alignments were performed to determine nucleotide and amino acid sequence similarities. To determine the phylogenetic relationships, the 260-bp hypervariable region of the F gene sequence obtained in this study was compared with the corresponding regions of representative published class II NDV sequences available in GenBank. Alignment and comparison of the nucleotide and amino acid sequences were performed using Clustal W in MEGA 5.0 (Tamura et al. ). Maximum likelihood (ML) tree was estimated using the best-fit general time reversible (GTR) model of nucleotide substitution with gamma distributed rate variation among sites and a heuristic SPR branch—swapping search available in PhyML version 3.0 (Guindon and Gascuel ). A bootstrap resampling process (100 replicates) was employed to assess the robustness of individual nodes of phylogeny. […]

Pipeline specifications

Software tools Clustal W, MEGA, PhyML
Application Phylogenetics
Organisms Avian avulavirus 2, Gallus gallus
Diseases Newcastle Disease
Chemicals Amino Acids