TABLE 2.
Nucleotide differences between the parental VR-2332 isolate and the full-length cDNA clone
| Nucleotide position within VR-2332 genomea | “Consensus” nucleotide of North American isolatesb | Nucleotide in cDNA clone | Change |
|---|---|---|---|
| 259 | G | A | Silent |
| 1075 | C | T | Silent |
| 5520 | C | T | Y → I |
| 5611 | T | A | Silent |
| 6854 | G | A | D → N |
| 6967 | T | C | Silent |
| 7555 | T | C | Silent |
| 10644 | T | C | Silent |
| 13788 | T | C | Silent |
| 15314 | G | T | Silent |
| 15318 | T | C | Silent |
Nucleotide positions within the VR-2332 genome are based on GenBank accession numbers AF094475 and PRU87392 (26,30).
After final assembly in pOK12, the infectious clone (15.4-kb fragment flanked by XhoI and XbaI sites in pOK12; Fig. 1) was sequenced in total (GenBank accession number AY150564). This sequence was compared to a “consensus” sequence of previously published full-length sequences of North American isolates (GenBank accession numbers PRU87392, AF066183, AF176348, AF046869, and AF159149) (2,3,26,38,41). In total, 11 nucleotide differences were observed, as shown in the table. The mutations at nucleotide positions 259, 15314, and 15318 were introduced intentionally to create BstZ17I and HpaI restriction sites, respectively. The sites were used for cloning purposes (see Fig. 1). Furthermore, the BstZ17I site was used as a genetic marker for the cloned virus.