Multiple orthoflaviviruses secrete sfRNA in mosquito saliva to promote transmission by inhibiting MDA5-mediated early interferon response

Numerous orthoflaviviruses transmitted through the bites of different mosquito species infect more than 500 million people annually. Bite-initiated skin infection represents a critical and conserved step in transmission and a deeper understanding of this process will promote the design of broad-spectrum interventions to address diverse orthoflavivirus health threats. Here, we identify and characterize a transmission-enhancing viral factor in mosquito saliva that is shared across orthoflaviviruses. Saliva of West Nile virus-infected Culex and Zika virus-infected Aedes contains a viral non-coding RNA, subgenomic orthoflaviviral RNA (sfRNA), within lipid vesicles distinct from virions. Higher concentration of sfRNA in infectious saliva positively correlates with infection intensity in human cells and skin explants. Early sfRNA delivery into transmission-relevant skin cell types and human skin explant demonstrate that sfRNA is responsible for the infection enhancement. Co-inoculation of sfRNA in a mouse model of transmission enhanced skin infection and worsened disease severity, evidencing the role of salivary sfRNA as a transmission-enhancer. Mechanistically, salivary sfRNA attenuates early interferon response in human skin cells and skin explants by altering MDA5-mediated signaling. Our results, derived from two distinct orthoflaviviruses and supported by prior studies, establish salivary sfRNA as a pan-orthoflavivirus transmission-enhancing factor driven by a conserved viral non-coding RNA.