ts53 virus has a substitution mutation from U to C at the nucleotide position of 701 in the PA gene. This substitution introduces an amino acid change from wild-type Leu 226 to Pro 226 and gives a defect in the viral genome replication process [48] . However, under the permissive temperature, the level of viral genome replication is no difference between wild-type and ts53 [47] . To discriminate the genome of wild-type and that of ts53, total RNA was reverse-transcribed by reverse transcriptase (TOYOBO) with PA-895-rev (59-TTAATTTTAAGGCATC-CATCAGCAGG-39), which is complementary to the segment 3 positive sense RNA. The cDNA was amplified by PCR using primers, PA-895-rev and PA-695-cut (59-TCTCCCGCCA-AACTTCTCAGGCC-39) partially corresponding to segment 3 positive sense RNA between nucleotide sequence positions 678 to 700 except for nucleotide positions 696 and 697. Since segment 3 of ts53 has a substitution mutation from U to C at the nucleotide position of 701, the PCR product derived from wild-type was digested by Stu I but not that from ts53. After PCR reactions, PCR products were digested with Stu I and separated through PAGE. Large and small fragments derived from ts53 and wild-type viruses were 220 and 199 base pairs, respectively. DNA was stained with GelRed (BIOTIUM) and visualized by UV illumination.