Exchanging the stem-loop A promoter of Zika virus with the corresponding region of tick-borne encephalitis virus generates a chimeric virus that replicates in vertebrate and mosquito cells

The 5' end of the orthoflavivirus genome contains a stem-loop structure, known as stem-loop A (SLA), which interacts with the viral polymerase (the NS5 protein) and functions as the promoter during RNA replication. Previously, we replaced the SLA of Zika virus (ZIKV; a mosquito/vertebrate orthoflavivirus) with the corresponding region of Long Pine Key virus (a dual-host-associated insect-specific orthoflavivirus), producing a chimeric virus capable of replicating in both mosquito and vertebrate cells. Here, we investigated whether additional chimeric viruses could be created by replacing the SLA of ZIKV with those of other orthoflaviviruses that are maintained in different transmission cycles, namely: tick-borne encephalitis virus (TBEV; a tick/vertebrate orthoflavivirus), Culex flavivirus (a classical insect-specific orthoflavivirus), Modoc virus (a rodent-associated vertebrate-specific orthoflavivirus), and Rio Bravo virus (a bat-associated vertebrate-specific orthoflavivirus). Exchanging the SLA of ZIKV with that of TBEV produced a chimeric virus capable of replicating in mosquito and vertebrate cells, whereas the other SLA replacements did not yield infectious virus. The chimeric virus replicated more slowly, reached lower titers, and produced smaller plaques than wild-type ZIKV in vertebrate cells. The chimeric virus also exhibited reduced fitness in mosquito cells. These findings demonstrate that replacement of the SLA of ZIKV with the corresponding region of TBEV produces a chimeric virus that replicates in both mosquito and vertebrate cells, revealing that ZIKV NS5 is able to recognize and bind to the TBEV promoter, rendering it active. This study highlights the importance of SLA-polymerase compatibility in flavivirus replication and provides insight into the molecular basis of host adaptation.