The existence of cell-to-cell transmission pathway gives a caution when NA inhibitors are used, because NA inhibitors may not be sufficient to completely block the spread of influenza Figure 6 . Influenza viruses can not re-infect previously infected cells. (A) A method for determination of the amount of segment 3 genome derived from ts53 and wild-type. Total RNA was reverse-transcribed with the primer PA-895-rev, which is complementary to the segment 3 positivesense RNA. The cDNA was amplified by PCR using primers, PA-895-rev and PA-695-cut partially corresponding to segment 3 positive sense RNA between the nucleotide sequence positions 678 to 700 except for 696 and 697, which are shown in red letters. 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 could be digested by Stu I but not that from ts53. Then, PCR products were digested with Stu I and separated through 8% PAGE. (B) Detection of the genome of the segment 3 derived from ts53 or wild-type. At 3 hours post superinfection of wild-type virus, total RNA was extracted, and semi-quantitative RT-PCR was performed. Subsequently, the amplified DNA products were digested with Stu I and separated through 8% PAGE. Large and small fragments derived from ts53 and wild-type viruses were 220 and 199 base pairs, respectively. The relative amount of wild-type segment 3 to that at 0 hour in the absence of oseltamivir phosphate was shown in the graph. Error bars indicate S.D. from 3 independent experiments. White bar, in the absence of oseltamivir phosphate; black bar, in the presence of oseltamivir phosphate. doi:10.1371/journal.pone.0028178.g006 virus in local microenvironments. Since this cell-to-cell transmission pathway exists, development of antiviral therapeutic strategies in addition to NA inhibitors is highly recommended.