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Protocol publication

[…] r diagnostic purposes by veterinarians from farms they provide care for through the period of 2006 to 2010. As those specimens were collected as part of the routine animal care, no animal care and use committee approval was sought. Five isolated viruses were included in this analysis (). All samples were tested for the presence of influenza A viruses by detection of M gene using one-step RT-PCR kit (Qiagen, Valencia, CA). Samples were subtyped using specific primers (). Comparative analysis of HA genes for all H5N1 viruses was carried out and compared with the available sequences using the NCBI influenza virus resource for both avian and human H5N1 viruses. Sequences were analyzed using the BioEdit program . Sequences were aligned with ClustalW . Phylogenetic analyses were carried out using MEGA version 4.0.2, with the neighbor-joining method, Poisson correction . Bootstrap values (1000 replications) are indicated on the tree., The antiviral susceptibilities of H5N1 influenza viruses were determined by fluorescence-based NA enzyme inhibition assay using 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA; Sigma, St. Louis, MO) at a final concentration of 100 µM as a substrate. The IC50 was defined as the concentration of NA inhibitor necessary to reduce NA activity by 50% relative to that of a reaction mixture containing virus but no inhibitor. Full-length NA and matrix (M2) proteins were sequenced to identify molecular markers of resistance., From March 2006 to Decem […]

Pipeline specifications

Software tools BioEdit, Clustal W, MEGA
Databases NCBI Influenza Virus Resource
Organisms Homo sapiens
Diseases Infection, Influenza, Human, Virus Diseases, Wiskott-Aldrich Syndrome