Discovery of Novel Alphacoronaviruses in European Rodents and Shrews

Tsoleridis, Theocharis; Onianwa, Okechukwu; Horncastle, Emma; Dayman, Emma; Zhu, Miaoran; Danjittrong, Taechasit; Wachtl, Marta; Behnke, Jerzy M.; Chapman, Sarah; Strong, Victoria; Dobbs, Phillipa; Ball, Jonathan K.; Tarlinton, Rachael E.; McClure, C. Patrick

Total RNA, from approximately one cubic millimetre sections of liver or intestinal tissue samples, was extracted using the GenEluteâ„¢ 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 [12] or a previously published primer set targeting a conserved region of the ORF1ab CoV polymerase gene [13] . 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 [13] . 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 [14] . 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. Total RNA, from approximately one cubic millimetre sections of liver or intestinal tissue samples, was extracted using the GenEluteâ„¢ 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 [12] or a previously published primer set targeting a conserved region of the ORF1ab CoV polymerase gene [13] . 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 [13] . 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 [14] . Codon-constrained nucleotide sequences were aligned using ClustalW and maximum