Figure 1.

Comparison of viral growth and nucleotide sequences. Each strain was inoculated into MNA (a,c,e) or DF-1 cells (f) at a multiplicity of infection (M.O.I.) of 0.05. Growth curves of MNA cells were compared between HEP and recombinant HEP (rHEP) (a), cloned HEP (cHEP) and cHEP-M(D80N) (c), and rHEP and rHEP-M(D80N) (e). In DF-1 cells, the growth curves were compared with HEP, rHEP, and rHEP-M(D80N) (f). For viral titration, antigen-positive foci were counted under a fluorescence microscope and calculated as focus-forming units (FFU) per milliliter. The mean viral titer and standard deviation (S.D.) were calculated from two or three independent experiments. Significant differences are indicated (*: p < 0.05, **: p < 0.01, ***: p < 0.001) after two-way analysis of variance (ANOVA) followed by Tukey’s test. Nucleotide sequences from the original HEP strain and the supernatant of MNA cells infected with HEP at 2 days post-infection (d.p.i.) (b) and those from cHEP and cHEP-M(D80N) (d) were compared. The sequences of these strains were determined and compared using GENETYX Ver.15 (GENETYX, Tokyo, Japan) and a Sequence Scanner (Thermo Fisher Scientific, Waltham, MA, USA). The arrowhead points to nucleotide position 238 (amino acid position 80) in the M protein. Black, orange, and red arrowheads indicate guanine, adenine, and a mixture of adenine and guanine, respectively. HEP: high-egg-passage Flury laboratory strain.