Conventional plaque assays rely on the use of overlays to restrict viral infection allowing the formation of distinct foci that grow in time as the replication cycle continues leading to counta-ble plaques that are visualized with standard techniques such as crystal violet, neutral red or immunolabeling. This classical approach takes several days until large enough plaques can be visualized and counted with some variation due to subjectivity in plaque recognition. Since plaques are clonal lesions produced by virus-induced cytopathic effect, we applied DNA fluores-cent dyes with differential cell permeability to visualize them by live cell imaging. We could observe different stages of that cytopathic effect corresponding to an early wave of cells with chromatin-condensation followed by a wave of dead cells with membrane permeabilization within plaques generated by different animal viruses. This approach enables an automated plaque identification using image analysis to increase single plaque resolution compared to crystal violet counterstaining and allows its application to plaque tracking and plaque reduction assays to test compounds for both antiviral and cytotoxic activities. This fluorescent real-time plaque assay sums to those next-generation technologies by combining this robust classical method with modern fluorescence microscopy and image analysis approaches for future applica-tions in virology.

Zika virus (ZIKV) is a mosquito-born flavivirus which human infection became relevant dur-ing recent outbreaks in Latin America, due to its unrecognized association with fetal neurologi-cal disorders. Currently there are no approved effective antivirals or vaccines for treatment or prevention of ZIKV infections. Amantadine and rimantadine are approved antivirals used against susceptible influenza A virus infections, that have been shown to have antiviral activity against other viruses, such as dengue virus (DENV). Here, we report the in vitro effectiveness of both amantadine and rimantadine hydrochlorides against ZIKV replication, resulting in a dose-dependent reduction in viral titers of a ZIKV clinical isolate and two different ZIKV refer-ence strains. Additionally, we demonstrate similar in vitro antiviral activity of these drugs against DENV-1 and yellow fever virus (YFV), although at higher drug concentrations for the later. ZIKV replication was inhibited at drug concentrations well below cytotoxic levels of both compounds, as denoted by the high selectivity indexes obtained with the tested strains. Further work is absolutely needed to determine a potential clinical use of these antivirals against ZIKV infections, but our results suggest the existence of a highly conserved mechanism across fla-vivirus, susceptible to be blocked by modified more specific adamantane compounds.

Arthropod-borne viruses belonging to the flavivirus genus possess an enormous relevance in public health. Neotropical non-human primates (NPs) have been proposed to be infected more frequently with flaviviruses due to their arboreal and diurnal habits, their genetic similarity to humans and their relative closeness to humans. However, the only known flavivirus in America that is maintained by sylvatic cycles involving NPs is Yellow Fever virus (YFV), and the NPs role as potential hosts of flaviviruses is still unknown. Here, we examined flavivirus exposure in 86 free range and captive NPs of Costa Rica to evaluate their involvement in flavivirus transmission cycles and their potential as flavivirus hosts. We used a highly-specific micro plaque reduction neutralization test (micro-PRNT) to determine the presence of antibodies against YFV, Dengue virus 1-4 (DENV), Zika virus, West Nile Virus (WNV) and Saint Louis Encephalitis virus (SLEV). We found evidence of seropositive NPs to DENV-1 8.2% (homotypic –3/86, heterotypic – 4/86), SLEV 15.1% (homotypic – 10/86, heterotypic – 2/86), WNV 2.3% (homotypic – 2/86) and 8.1% (7/86) undetermined Flavivirus species. No antibodies against YFV or ZIKV were found. This work provides compelling serological evidence of exposure in NPs of flaviviruses associated with urban cycles, i.e. DENV, and confirms decades of circulation of SLEV in the same environments. Also, the range of years of sampling and the socioeconomic region was statistically significant for the presence of Dengue and Flavivirus undetermined seropositive individuals, respectively. Both the years and socioeconomic regions with greater seroprevalence coincide with the years and socioeconomic regions with high numbers of Dengue human cases for the country. Our work suggests bidirectional? circulation of different flaviviruses between humans and wildlife with public health importance and underscores the necessity of further surveillance for flaviviruses in the humans/wildlife interface in Central America.

Arboviruses have two ecological transmission cycles, sylvatic and urban. For some, the sylvatic cycle has not been thoroughly described in America. To study the role of wildlife in a putative sylvatic cycle, we sampled free-ranging bats and birds in two arbovirus endemic locations and analyzed them using molecular, serological, and histological methods. No current infection was detected, and no significant arbovirus-associated histological changes were observed. Neutralizing antibodies were detected against selected arboviruses. In bats, positivity in 34.95% for DENV-1, 16.26% for DENV-2, 5.69% for DENV-3, 4.87% for DENV-4, 2.43% for WNV, 4.87% for SLEV, 0,81% for YFV, 7.31% for EEEV, and 0.81% for VEEV was found. Antibodies against ZIKV were not detected. In birds, PRNT results were positive against WNV in 0.80%, SLEV in 5.64%, EEEV in 8.4%, and VEEV in 5.63%. An additional retrospective PRNT analysis was performed using bat samples from three additional DENV endemic sites resulting in a 3.27% prevalence for WNV and 1.63% for SLEV. Interestingly one sample resulted unequivocally WNV positive confirmed by serum titration. These results suggest that free-ranging bats and birds are exposed to not currently reported hyperendemic-human infecting Flavivirus and Alphavirus, however, their role as reservoirs or hosts is still undetermined.