Primary viral infections (infections of non-immune individuals) are a major cause of morbidity and mortality in humans. Experimental and clinical data show that Natural Killer (NK) cells play an essential role in the control of many primary vira infections. The mechanisms of this control during the natural course of infection remain poorly defined. Many viruses relevant to human and animal health use a lympho- hematogenous (LH) route of spread whereby they breach epithelial surfaces and then spread stepwise through the regional draining lymph node (D-LN) and then the blood to produce a systemic disease. The Orthopoxvirus Ectromelia virus (ECTV), the agent of mousepox, is an outstanding model of natural infections and serves as the textbook example of viruses that disseminate through the LH route. We have previously shown that during the first two days after footpad ECTV infection of mousepox-resistant young C57BL/6 (B6) mice, activated mature NK cells accumulate in the D-LN. Importantly, we have also found that the mature NK cells that accumulate in the D-LN curb virus spread to the liver. This is critical for resistance to mousepox because mice succumb when their NK cells do not accumulate in the D-LN. Interestingly, B6 mice deficient in Toll Like Receptor 9 (TLR9) and its signaling adapter, MyD88 (MyD88) are highly susceptible to mousepox. Important for this application, mature NK cells do not accumulate in the D-LN of TLR9 and MyD88 deficient mice. The fundamental hypothesis in this application is that the accumulation of NK cells in the D-LN and their ability to curb virus spread is a major mechanism of defense against viruses that disseminate through the LH route. Thus, our major goals are to identify the molecular (Aim 1) and cellular (Aim 2) mechanisms that result in the specific accumulation of mature NK cells in the D-LN during ECTV infection with an emphasis on the role of the TLR9/MyD88 pathway and whether the virus subverts this process. Furthermore, we will determine whether comparable mechanisms of NK cell recruitment to the D-LN are operative during other viral infections and whether they contribute to decrease virus spread (Aim 3).