Whole Genome Sequencing of NVAV Strain

Oligonucleotide primers were designed using the MegAlign Clustal W program (DNASTAR Inc., Madison, WI) to obtain the whole genome of NVAV strain Te34 from cDNA prepared from total RNA, extracted from NVAV-infected Vero E6 cells and from lung tissue of the original wild-caught European mole (Table 2). The 5′– and 3′–termini of each segment were amplified using the 3′–Full RACE Core Set (Takara Bio Inc., Otsu, Japan). Nested or hemi-nested PCR was performed in 20 μL reaction mixtures, containing 250 μM dNTP, 2.5 mM MgCl₂, 1 U of Takara LA Taq polymerase (Takara) and 0.25 μM of each primer (Table 2). Initial denaturation at 94 °C for 2 min was followed by two cycles each of denaturation at 94 °C for 30 sec, two-degree step-down annealing from 46 °C to 38 °C for 40 sec, and elongation at 72 °C for 1 min, then 30 cycles of denaturation at 94 °C for 30 sec, annealing at 42 °C for 40 sec, and elongation at 72 °C for 1 min, in a GeneAmp PCR 9700 thermal cycler (Perkin-Elmer, Waltham, MA)34 (link). PCR products were separated, using MobiSpin S-400 spin columns (MoBiTec, Goettingen, Germany), and amplicons were sequenced directly using an ABI Prism 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA)34 (link).