To learn how quickly rZH501-M847-A-type virus accumulated in various organs of rZH501-M847-G-infected mice, we inoculated 25 mice i.p. with 10 5 PFU of rZH501-M847-G, and livers, spleens and brains were isolated every day through day 8 p.i. To eliminate the possible contamination by rZH501-M847-A in the samples, rZH501-M847-A was not used in the BSL-4 laboratory during the experiments. For each sample, the virus titers, production of RT-PCR products and susceptibilities of the RT-PCR products to Sac I digestion are summarized in Fig. 8A and the representative data concerning the Sac I digestion of the RT-PCR products are shown in Fig. 8B . Most samples carrying detectable levels of infectious viruses and many samples in which virus titers were below the detection limit yielded the RT-PCR products. Accumulation of rZH501-M847-A-type virus was detected in the liver of a single mouse as early as 2 days p.i. and rZH501-M847-A-type virus became the major virus population in livers, spleens and brains from day 5 to 8 p.i. We performed sequence analysis of some of the cloned RT-PCR products to firmly establish the accumulation of rZH501-M847-A-type virus in rZH501-M847-G-infected mice; the numbers of the clones used for sequence analysis and the samples were: 20 clones from mouse 8b brain, 10 clones from mouse 6c spleen, 10 clones from mouse 7c brain, and 10 clones from mouse 7c spleen (see Fig. 8A ). Consistent with the data of Sac I digestion of the RT-PCR products of these samples, all clones had A at M847. These data unambiguously demonstrated that rZH501-M847-Atype virus became the major virus population in rZH501-M847-G-infected mice. To know whether the phenomenon of emergence of a major population of rZH501-M847-A-type virus after infection with rZH501-M847-G-type virus also occurred in cell culture, rZH501-M847-G was independently passaged five times at a multiplicity of infection (moi) of 0.01 and 1 in both VeroE6 cells and MRC-5 cells. RT-PCR products, including M847, were generated from intracellular RNAs, extracted from cells infected with viruses at passage levels 1 and 5, and subjected to Sac I digestion. We did not observe the accumulation of rZH501-M847A-type virus after 5 passages of rZH501-M847-G in both cell types (data not shown), demonstrating that the rZH501-M847-A-type virus did not emerge after passages of rZH501-M847-G in cultured cells but it became the major virus population in mice that were infected with rZH501-M847-G.