Computational protocol: Intrahost Diversity of Feline Coronavirus: A Consensus between the Circulating Virulent/Avirulent Strains and the Internal Mutation Hypotheses?

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Protocol publication

[…] cDNA was synthesised using Random Primers (Invitrogen, Carlsbad, CA, USA) and M-MLV Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's protocols. The partial M gene (575 bp) was then amplified using 0.5 μM of both reverse (26925–26944 nt positions regarding strain FCoV UU47) and forward primers (26968–26292 nt positions regarding strain FCoV UU47) [] and GoTaq Green Master Mix 1X (Promega Corporation, Madison, WI, USA) as per the manufacturer's instructions. Amplicons were purified from agarose gels with Illustra (GE Healthcare, Buckinghamshire, UK) and submitted to bidirectional DNA sequencing with BigDye 3.1 (Applied Biosystems, Carlsbad, CA, USA), according to the manufacturer's protocols. Products were resolved using a 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA), and the chromatograms were analysed with Phred at http://asparagin.cenargen.embrapa.br/phph/. Positions with a quality score >20 were used to generate contiguous sequences with Cap-contig implemented in the software BioEdit 7.0.9.0 []. Those sequences were then submitted to BLAST/n at http://www.ncbi.nlm.nih.gov/BLAST to confirm the Amplicon identities.The M gene partial sequences and the putative amino acid sequences from each sample were aligned with homologous sequences from both FCoVs pathotypes retrieved from GenBank () with CLUSTAL/W in BioEdit 7.0.9.0 [], and a phylogenetic tree for the nucleotide sequences was generated with the neighbour-joining distance algorithm and the maximum composite likelihood model with 1,000 bootstrap replicates using MEGA 5.0 []. Amino acid alignment was used to search for previously reported pathotype-specific markers []. […]

Pipeline specifications

Software tools BioEdit, Clustal W, MEGA
Application Phylogenetics
Organisms Felis catus, Feline coronavirus, Feline infectious peritonitis virus
Diseases Feline Infectious Peritonitis