The present study showed the importance of the A nucleotide at M847 of wt RVFV in mouse virulence, whereas it is unclear whether this nucleotide also affects viral virulence in other animals. Like rZH501-M847-G, one wt RVFV isolate 763/70, which was obtained from an aborted bovine fetus in Zimbabwe in 1970 [28] , has G at M847 and carries Gly 277 [19] , while all the rest of the wt RVFV isolates thus far sequenced carry A at M847 and encode Glu277 [19] . No information is available as to the titers of 763/70 in different tissues of the aborted fetus and the viremia status of its mother. Although we reported that pregnant cows inoculated with ZH501 become febrile, have 2.3 to 4.0 log 10 PFU/ml of viremia, and undergo abortion [29] , the relationship between level of viremia and abortion in cattle has not been carefully examined yet. We observed that very low viremia can result in abortion in experimentally infected sheep with ZH501 (J. Morrill, unpublished data), and, hence, we speculate that placental tissue in sheep and probably cattle may be exquisitely sensitive to infection, and low titers of viremia can infect the placenta at very low levels and gain access to the fetus. Virulence of 763/70 in the bovine has not been experimentally tested and 763/70 differs from ZH501 at other genome regions. Accordingly, the role of G at M847 of 763/70 in virus virulence for bovine is unclear. Experimental infection of rZH501-M847-A and rZH501-M847-G into domestic animals will show whether the importance of the A nucleotide at M847 in viral virulence is limited to the mouse model. Figure 7 . Sac I digestion of the RT-PCR products. Five-week-old female CD-1 mice were infected i.p. with 10 5 PFU of rZH501-M847-G or rZH501-M847-A. Total RNAs from 3 livers at 4 day p.i., 3 spleens at day 6 p.i., 3 kidneys at day 6 p.i., 3 brains at day 6 p.i., and 3 brains at day 7 p.i. were collected and subjected to RT-PCR analysis. In the samples where RT-PCR products were obtained, RT-PCR products were digested with Sac I and subjected to agarose gel electrophoresis. As controls, PCR products that were obtained from plasmid encoding M segment of rZH501-M847-A were digested with Sac I or incubated in the absence of Sac I (Control). doi:10.1371/journal.pone.0009986.g007 Figure 8 . Accumulation of viruses carrying the genotype of rZH501-M847-A in rZH501-M847-G-infected mice. Five-week-old female CD-1 mice were infected i.p. with 10 5 PFU of rZH501-M847-G and liver, spleen and brain were collected every day from day 1 to day 8 p.i. Virus titer in each sample was determined by a plaque assay. RT-PCR products were obtained from total RNAs in these samples and then subjected to Sac I digestion. (A) Color and number in each box represent the genotype of major viruses and the virus titer in Log 10 PFU/g of tissue, respectively, in each mouse. Color legend: G = genotype of rZH501-M847-G. A = genotype of rZH501-M847-A. G = A = a mixture of similar amounts of rZH501-M847-G genotype and rZH501-M847-A genotype. G.A = viruses carrying the rZH501-M847-G genotype were more abundant than those carrying the rZH501-M847-A genotype. A.G = viruses carrying the rZH501-M847-A genotype were more abundant than those carrying the rZH501-M847-G genotype. Not decided = the major genotype of viruses was not determined due to weak RT-PCR signals. Not detected = RT-PCR products were not obtained. (B) Agarose gel electrophoresis of the RT-PCR products that underwent SacI digestion. Only representative samples are shown. The mouse identification number in the gel is shown in (A). Control = PCR products that were obtained from plasmid encoding M segment of rZH501-M847-A were digested with Sac I or incubated in the absence of Sac I. N = Not detected. W = Not decided. doi:10.1371/journal.pone.0009986.g008