Tumor cells were injected (s.c.) into C3H or BALB/c mice, respectively. Mice with established tumors were treated by peritumoral injections of CpG-Stat3 siRNA, CpG-Luc siRNA, CpG alone or PBS every other day, starting 7d after challenge with 1×10 5 tumor cells. Statistically significant differences between the CpG-siRNA-treated groups are indicated by asterisks. (e) C57BL/6.CEA mice were challenged s.c. with 1×10 5 of MC38.CEA cells and treated as described above using CpG-Stat3 siRNA or CpG-Luc siRNA starting from d 11 after tumor challenge, P < 0.0001 by two-way ANOVA (n = 4 for each group). (f) Systemic treatment with CpG-Stat3 siRNA reduces Stat3 expression in DCs within TDLNs (cervical). Samples pooled from 6 mice/group were analyzed by real-time PCR. Shown is the average level of Stat3 expression in CpG-Stat3 siRNA-treated mice from one of two independent experiments analyzed in triplicates ± SEM, relative to control CpGscrambled RNA, which was set as 100%.  Rapid Accumulation of Virulent Rift Valley Fever Virus in Mice from an Attenuated Virus Carrying a Single Nucleotide Substitution in the M RNA BACKGROUND: Rift Valley fever virus (RVFV), a member of the genus Phlebovirus within the family Bunyaviridae, is a negative-stranded RNA virus with a tripartite genome. RVFV is transmitted by mosquitoes and causes fever and severe hemorrhagic illness among humans, while in livestock it causes fever and high abortion rates. METHODOLOGY/PRINCIPAL FINDINGS: Sequence analysis showed that a wild-type RVFV ZH501 preparation consisted of two major viral subpopulations, with a single nucleotide heterogeneity at nucleotide 847 of M segment (M847); one had a G residue at M847 encoding glycine in a major viral envelope Gn protein, while the other carried A residue encoding glutamic acid at the corresponding site. Two ZH501-derived viruses, rZH501-M847-G and rZH501-M847-A, carried identical genomic sequences, except that the former and the latter had G and A, respectively, at M847 were recovered by using a reverse genetics system. Intraperitoneal inoculation of rZH501-M847-A into mice caused a rapid and efficient viral accumulation in the sera, livers, spleens, kidneys and brains, and killed most of the mice within 8 days, whereas rZH501-M847-G caused low viremia titers, did not replicate as efficiently as did rZH501-M847-A in these organs, and had attenuated virulence to mice. Remarkably, as early as 2 days postinfection with rZH501-M847-G, the viruses carrying A at M847 emerged and became the major virus population thereafter, while replicating viruses retained the input A residue at M847 in rZH501-M847-A-infected mice. CONCLUSIONS/SIGNIFICANCE: These data demonstrated that the single nucleotide substitution in the Gn protein substantially affected the RVFV mouse virulence and that a virus population carrying the virulent viral genotype quickly emerged and became the major viral population within a few days in mice that were inoculated with the attenuated virus. Rift Valley fever virus (RVFV), a member of the genus Phlebovirus within the family Bunyaviridae, causes periodic outbreaks among livestock and humans in sub-Saharan African countries [1] . RVFV infection results in high mortality and abortion rates in domestic ruminants with severe hepatic diseases. It also causes an acute febrile myalgic syndrome, a hemorrhagic syndrome, ocular disease, and encephalitis in humans [2, 3] . Transmission of RVFV to humans is primarily mosquito-borne or due to direct contact with infected animal blood, tissues or products of abortion. Since the late 1970s, several major outbreaks of Rift Valley fever have occurred outside of sub-Saharan Africa, e.g., in Egypt [4] , Madagascar [5] , Saudi Arabia, and Yemen [2, 6] . The most recent outbreak was reported in Kenya and resulted in a high reported case-fatality ratio in infected humans [7] .