(A) Representative images of Vero cells infected with RV (top row) or Zika virus as control (bottom row). At 72hpi, cells were fixed and processed for immunofluorescence with anti-RV capsid antibody (RVcap) or Zika virus antibody (Zika4G2), and then FISH was performed using probes to positive strand (+) or negative strand (-) RV RNA. Negative strand RV RNA difficult to visualize at low-power magnification, and required quantification within cell borders defined by wheat germ agglutinin staining with results in panel B. (B) In Vero cells, negative strand RV RNA is detected in strongly infected cells. Infection strength determined by intensity of RV capsid immunofluorescence staining and positive strand RV RNA (RVcap/(+) 2/3 indicates robust infection, RVcap/(+) 1 indicates weak infection). ZIKVinf = Zika virus infected control. (C) In microglia co-cultures, positive strand RV RNA detected in cells with RV capsid immunopositivity (RVcap_pos). RVinf = RV infected. RVHI = heat-inactivated RV. (D) In microglia co-cultures, negative strand RV RNA quantification not significantly different between mock, heat-inactivated RV (RVHI), or RV- infected conditions (RVinf), including cells with weak positive-strand RV RNA (RVinf, (+)<8) or cells with stronger positive-strand RV RNA (RVinf, (+)>=8). Two biological replicates (bHR60 and bHR61), n indicates number of cells counted.