Although we have demonstrated the importance of single nucleotide substitution at M847 of Gn protein for wt RVFV mouse virulence, further studies are required to determine how this single nucleotide substitution affected RVFV's virulence. Past studies showed that the gain-of-positive-charge mutations in the flavivirus E protein [21, 22, 23] and the alphavirus E2 protein [24] can enhance binding to negatively charged glycosaminoglycans (GAG) and increase the efficiency of virus uptake into cultured cells. However, the virulence of the variant viruses in mice is reduced, as these variant viruses are rapidly cleared from the circulatory system [22, 24, 25, 26] . Probably, the gain-of-positivecharge mutations in the envelope protein facilitates viral adherence to GAGs present in blood cells and subsequent reticuloendothelial system-mediated virus elimination from the circulation. Likewise, rZH501-M847-A, which had a negatively charged Glu277 of the Gn envelope protein, was highly virulent for mice, while rZH501-M847-G carrying an uncharged Gly277 was substantially less virulent (Fig. 3) . Furthermore, rZH501-M847-A-infected mice showed higher viremia titers than rZH501-M847-G-infected mice at 1-3 days p.i. (Fig. 4) , but rZH501-M847-G replicated at a higher titer than did rZH501-M847-A in MRC-5 cells (Fig. 2) . Accordingly, it is possible that rZH501-M847-G binds efficiently to GAGs in the circulation, resulting in low levels of systemic infection, whereas rZH501-M847-A carrying Glu277 binds to GAGs less efficiently, resulting in high titers of viremia, efficiently establishes a systemic infection that involves the CNS, and kills the infected mice. The attenuated virus, MP-12, developed by serial passage of the wt RVFV ZH548 strain in the presence of 5-fluorouracil [27] , carries A at M847 and encodes Glu277, suggesting that the presence of Glu277 in Gn does not always make RVFV virulent. The amino acid sequence of the M segment of MP-12 differed from that of rZH501-M847-A by 5 amino acids. We suspect that a combination of some or all of these mutated amino acids in the M segment contributed to the attenuation of MP-12.