Bats Possess Unique Variants of the Antiviral Restriction Factor Tetherin

Joshua A. Hayward; Mary Tachedjian; Adam Johnson; Tamsin B. Gordon; Jie Cui; Glenn A. Marsh; Michelle L. Baker; Lin-Fa Wang; Gilda Tachedjian

To amplify tetherin nucleotide sequences from bat cDNA, polymerase chain reaction (PCR) assays were performed using cDNA generated from P. alecto and M. ricketti spleen tissue, and M. macropus and M. schreibersii kidney cell lines, using various combinations of forward and reverse primers (Table 3A) . Primers were designed using the tetherin predictions identified in the contig and SRA analyses. Primers were designed to bind to the 5' and 3' untranslated regions of bat cDNA such that the full CDS could be amplified. Bat capture, tissue collection and RNA extraction was conducted as previously reported (56) with the exception that RNAlater (Ambion) preserved spleen tissue from four male adult bats was pooled before tissue homogenisation and followed with total RNA extraction with the Qiagen RNeasy Mini kit with on-column extraction of genomic DNA with DNase I. Total RNA was reverse transcribed into cDNA with the Qiagen Omniscript reverse transcriptase according to the manufacturer's protocol with the exception that the reaction contained 100 ng/μl total RNA, 1 μM oligo-dT18 (Qiagen) and 10 μM random hexamers (Promega).