The recent emergence of Zika virus (ZIKV) in the Americas coincident with increased caseloads of microcephalic infants and Guillain-Barre syndrome has prompted a flurry of research on ZIKV. Much of the research is difficult to compare or repeat because individual laboratories use different virus isolates, growth conditions, and quantitative assays. Here we obtained three readily available contemporary ZIKV isolates and the prototype Ugandan isolate. We generated stocks of each on Vero mammalian cells (ZIKVmam) and C6/36 mosquito cells (ZIKVmos), determined titers by different assays side-by-side, compared growth characteristics using one-step and multi-step growth curves on Vero and C6/36 cells, and examined plaque phenotype. ZIKV titers consistently peaked earlier on Vero cells than on C6/36 cells. Contemporary ZIKV isolates reached peak titer most quickly in a multi-step growth curve when the amplifying cell line was the same as the titering cell line (e.g., ZIKVmam titered on Vero cells). Growth of ZIKVmam on mosquito cells was particularly delayed. These data suggest that the ability to infect and/or replicate in insect cells is limited after growth in mammalian cells. In addition, ZIKVmos typically had smaller, more homogenous plaques than ZIKVmam in a standard plaque assay. We hypothesized that the plaque size difference represented early adaptation to growth in mammalian cells. We plaque purified representative-sized plaques from ZIKVmos and ZIKVmam. ZIKVmos isolates maintained the initial phenotype while plaques from ZIKVmam isolates became larger with passaging. Our results underscore the importance of the cells used to produce viral stocks and the potential for adaptation with minimal cell passages. In addition, these studies provide a foundation to compare current and emerging ZIKV isolates in vitro and in vivo.

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Functioning healthcare systems provide emergency medical care. Disparities exist in accessibility and availability of emergency care in low- and middle-income countries. We present a descriptive epidemiologic analysis of Emergency Department (ED) usage in a rural, indigenous Guatemalan population. San Lucas Tolimán is situated in central Guatemala. Hospital Parroquial de San Lucas offers emergency care to San Lucas Tolimán and surrounding villages. All ED visits between January 1st, 2016 and December 31st 2018 were recorded and analyzed. During the study period, 12,229 patient encounters occurred. Almost all patients identified as indigenous. Children comprised 43% of visits. Medical issues represented a majority (83%) of complaints. Respiratory (40%) and gastrointestinal disease (26%) were frequent presenting complaints. Almost all visits (83%) occurred during the day and evening hours. Trauma/surgical complaints were slightly more frequent at night. 93% of patients were discharged, while the rest were admitted or transferred. These data contribute to understanding of disease burden and emergency care needs and capacity in rural areas of low- and middle-income countries. This information may be used to inform local policy decisions, identify research priorities, and create training topics for local health care providers in Guatemala and other countries in this region.