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Bats are important natural reservoir hosts of a diverse range of viruses that can be transmitted to humans and have been suggested to play an important role in the Zika virus (ZIKV) transmission cycle. However, the exact role of these animals as reservoirs for flaviviruses is still controversial. To further expand our understanding of the role of bats in the ZIKV transmission cycle in Latin America, we carried out an experimental infection in wild-caught Artibeus lituratus bats and sampled several free-living neotropical bats across three countries of the region. Experimental ZIKV infection was performed in wild-caught adult bats (4 females and 5 males). The most relevant findings were hemorrhages in the bladder, stomach and patagium. Significant histological findings included inflammatory infiltrate consisting of a predominance of neutrophils and lymphocytes, in addition to degeneration in the reproductive tract of males and females. This suggests that bat reproduction might be at some level affected by ZIKV. Leukopenia was also observed in some inoculated animals. Hemorrhages, genital alterations, and leukopenia are suggested to be caused by ZIKV; however, since these were wild-caught bats, we cannot exclude other agents. Detection of ZIKV by qPCR was observed at low concentrations in only two urine samples in two inoculated animals. All other animals and tissues tested were negative. Finally, no virus-neutralizing antibodies were found in any animal. To determine ZIKV infection in nature, the blood of a total of 2056 bats was sampled for ZIKV detection by qPCR. Most of the sampled individuals belonged to the genus Pteronotus sp. (23%), followed by the species Carollia sp. (17%), Anoura sp. (14%), and Molossus sp. (13.7%). No sample of any tested species was positive for ZIKV by qPCR. These results together suggest that bats are not efficient amplifiers or reservoirs of ZIKV and may not have an important role in ZIKV transmission dynamics. Author summary: In previous works in 2008–2009, we found the presence of antibodies against flaviviruses, and viral RNA was detected in Neotropical chiropterans in Mexico, which led us to support the hypothesis that these animals could be reservoirs of flaviviruses. As controversial opinions have been exposed and based on a previous (2019) experimental ZIKV infection experiment conducted at Colorado State University using adult Artibeus males from a captive colony, in this work, we also experimentally infected adult Artibeus males complementarily adding females and using wild-caught animals instead of laboratory bats. We also monitored a diverse range of natural bat populations in Latin America for the presence of viral RNA against ZIKV in blood. A plaque reduction seroneutralization test was used for the detection of antibodies against ZIKV. Similar to the previous work, we found histopathological alterations in male testicles but also in the ovaries and oviducts of females, as well as gliosis and multifocal necrosis in pyramidal neurons and Purkinge cells of inoculated animals. Only two urine samples from inoculated animals showed viral RNA. Additionally, leukopenia and lymphoid follicular splenic hyperplasia were evidenced. In contrast to what was reported, no neutralizing antibodies against ZIKV were detected in any sample. Viral RNA within the blood was not present in any of the 2056 bat samples collected in French Guiana, Peru and Costa Rica and proceeding from 34 bat genera. These results together suggest that bats are not efficient amplifiers or reservoirs of ZIKV and might not have an important role in ZIKV transmission dynamics. [ABSTRACT FROM AUTHOR]