TABLE 1.
Nucleotide sequence analysis of HAV clones and constructs grown at different concentrations of blasticidin
| Virusa | Mutation position in the HAV genomeb |
Cell growth in blasticidin (μg/ml)c |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 5545 (nt) | 3Cpro (aa) | 6069 (nt) | 3Dpol (aa) | 6780 (nt) | 3Dpol (aa) | 7027 (nt) | 3Dpol (aa) | 1 | 5 | 20 | |
| HAV/7 | C | L | T | F | C | I | C | L | − | − | − |
| HAVvec9-Bsdd | C | L | T | F | C | I | C | L | + | +/− | − |
| HAVvec9-Bsd-5 | |||||||||||
| 4 clones | C | L | G | L | C | I | T | L | + | + | + |
| 2 clones | C | L | T | F | C | I | T | L | + | +/− | − |
| HAVvec9-Bsd-20 | |||||||||||
| 5 clones | T | L | G | L | T | I | T | L | + | + | + |
| 1 clone | T | L | T | F | T | I | T | L | + | +/− | − |
| HAVvec9-Bsd-6069 | C | L | G | L | C | I | C | L | + | + | + |
| HAVvec9-Bsd-7027 | C | L | T | F | C | I | T | L | + | +/− | − |
| HAVvec9-Bsd-6069 + 7027 | C | L | G | L | C | I | T | L | + | + | + |
The HAV/7 nucleotide sequence was obtained from the infectious HAV cDNA in pT7HAV. The sequences of six clones of HAVvec9-Bsd, -5, and -20 were obtained by RT-PCR and automatic sequencing of the whole viral genome. HAVvec9-Bsd-6069, HAVvec9-Bsd-7027, and HAVvec9-Bsd-6069 + 7027 were rescued from FRhK4 cells transfected with T7 polymerase in vitro transcripts from pHAVvec9-Bsd constructs containing T6069G, C7027T, and both T6069G and C7027T mutations, respectively. The nucleotide sequence of the rescued viruses was obtained by RT-PCR and automatic sequencing. Mutations were identified at nucleotides only at positions 5545, 6069, 6780, and 7027.
HAV genome nucleotide (nt) sequence at positions 5545 in the protease (3Cpro) and 6069, 6780, and 7027 in the polymerase (3Dpol). Corresponding amino acid (aa) residues at the codons defined by the nucleotide positions. Changes in nucleotide and amino acid positions are indicated by bold characters.
After infection, cells were selected with 1 or 5 or 20 μg/ml blasticidin, and presence (+) of blasticidin-resistant colonies, absence (-) of surviving cells, or presence of few surviving cells that did not form colonies (+/−) was determined at 7 dpi under the microscope.
Six clones were sequenced, and all contained the same nucleotides at positions 5545, 6069, 6780, and 7027.