infection One-step growth curve analysis showed significantreductioninfection One-step growth curve analysis showed significantreduction

infection (dpi), presence of virus encoded eGFP fluorescence inuntreated cells but not in gB-GNP treated cells indicated that allthree types of gB-GNP successfully blocked CMV infection(Fig. 2A–H). The cytopathogenic effects evident in bright-fieldimages in the absence of gB-GNP treatment (Fig. 2A) were notapparent in the treated cells (Fig. 2B–D). Also, when gB-GNPop wereadded to AD169 infected HF at one dpi, only primary infected cellsshowed eGFP fluorescence in gB-GNPop treated cells at 3 dpi,whereas both the primary and secondary infected cells showed fluorescencein untreated cells (Fig. 2I–P). This was true for high (1.0)(Fig. 2M, N) as well as low (0.1) (Fig. 2O, P) MOI. Thus, gB-GNPopblocked AD169 spread as well as primary infection in HF. Similarresults were obtained for the Towne strain of human CMV as well(extended data 1) however, cytopathogenic effects induced by simiancytomegalovirus (SCMV) infection of HF were not inhibited by gBGNPoptreatment (extended data 2). Also, HSV-1 infection of HFwas not abolished due to gB-GNPop treatment (extended data 3),consistent with the specificity of gB-GNPop for CMV gB. The fluorescencein HSV infected cells did reduce slightly upon gB-GNPoptreatment suggesting some non-specific interference with HSV particles(extended data 3). One-step growth curve analysis showed significantreduction in CMV (Towne-BAC) titers when treated withgB-GNPop (Fig. 2 Q) and the final viral yields were at least 10,000fold less than virus yields from untreated cells. We also tested clinicalstrain of human CMV derived from FIX-BAC that can infectendothelial cell lines19. HMEC-120 cells infected with FIX virusshowed a significant reduction in the expression of viral IE1 proteinin gB-GNPop treated cells compared to untreated cells, probed byimmunofluorescence at 8 hours post infection (hpi) (extended data 4A, D). Viral encoded eGFP showed a similar pattern of reducedfluorescence in treated cells (extended data 4 C, F). Thus gBGNPopsuccessfully blocked entry of a clinical strain of HCMV inthese cell types making this approach more clinically relevant. To testwhether these conjugated nanoparticles affect cell viability, HF weretreated with the working concentration of gB-GNP (50 ng/ml antibodytotal) (data not shown) as well as double the concentration(100 ng/ml antibody total) at the time of infection and cell viabilitywas recorded at 8 hpi and also at 5 dpi using trypan blue exclusionassay (Fig. 3). All three types of gB-GNP showed little to no toxicity inHF at this time. Only gB-GNPst showed some level of cytotoxicity,which did not develop until 5 dpi (Fig. 3B). The results show that gBGNPare generally not cytotoxic at the concentration at which theyblock HCMV infection.To visualize the binding of gB-GNP to virus particles, purifiedAD169 virions were incubated with gB-GNP on ice for 30 minutes,