Computational protocol: Discovery of Novel Alphacoronaviruses in European Rodents and Shrews

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Protocol publication

[…] In total, 899 tissue samples from 813 wild rodents and shrews from the East Midlands region of the United Kingdom and the Mazury Lake District region of Poland (, ) were collected either as part of routine pest control, as a consequence of predation (Felis catus) or for other studies []. The samples from the United Kingdom were collected between 2008 and 2015 and the study was approved by the University of Nottingham Internal Ethics Committee (Nottingham, UK). The study for the Polish samples was approved by the Ethics Commission for Experiments on Animals of the M. Nencki Institute of Experimental Biology of the Polish Academy of Sciences (Warszawa, Poland). The rodent species included: house mouse (Mus musculus), brown rat (Rattus norvegicus), field vole (Microtus agrestis), bank vole (Myodes glareolus), wood mouse (Apodemus sylvaticus) and shrew (Sorexaraneus). A necropsy was performed and tissue samples were stored at −70 °C in ethanol (Polish liver specimens), at −70 °C in RNAlater (Sigma Aldrich, St. Louis, MO, USA; UK liver and gut specimens) or at −20 °C without additives (UK liver specimens collected in 2008).Total RNA, from approximately one cubic millimetre sections of liver or intestinal tissue samples, was extracted using the GenElute™ Mammalian Total RNA Miniprep Kit (Sigma Aldrich) and used as template in reverse-transcription PCRs using in-house designed primers targeting the rodent glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene (F: 5’-CCATCTTCCAGGAGCGAGA-3’, R: 5’-GCCTGCTTCACCACCTTCT-3’), cytochrome b gene [] or a previously published primer set targeting a conserved region of the ORF1ab CoV polymerase gene []. Extra sequence for ORF1ab was acquired by using an in-house primer targeting an upstream conserved region (F: 5’-AATCTTAAGTATGCTATTAGTGG-3’) in combination with the previously described reverse primer []. PCR products of expected size were subject to Sanger sequencing (Source Bio Science, Nottingham, UK) and sequence similarity to Genbank database sequences was determined using BLASTn. Coronavirus ORF1ab and rodent cytochrome b reference sequence sets were downloaded from Genbank and used alongside sequences obtained in this study for phylogenetic analysis using the Molecular Evolutionary Genetics Analysis version 6 (MEGA6) software []. Codon-constrained nucleotide sequences were aligned using ClustalW and maximum likelihood phylogenetic trees (utilising a GRT with invariant sites (G+I) model of evolution) were generated, with robustness assessed using bootstrap resampling (1000 pseudoreplicates). CoV sequences generated in this study have been deposited in the Genbank database under accession numbers KU739070-KU739074. […]

Pipeline specifications

Software tools BLASTN, MEGA, Clustal W
Application Phylogenetics
Organisms Rattus norvegicus, Sorex araneus
Chemicals Nucleotides