Characterization of Mayaro virus (strain BeAn343102) biology in vertebrate and invertebrate cellular backgrounds

Abstract

Mayaro virus (MAYV) is an emerging New World alphavirus (genus Alphavirus, family Togaviridae) that causes acute multiphasic febrile illness, skin rash, polyarthritis and occasional severe clinical phenotypes. The virus lifecycle alternates between invertebrate and vertebrate hosts. Here we characterize the replication features, cell entry, lifecycle and virus-related cell pathology of MAYV using vertebrate and invertebrate in vitro models. Electron-dense clathrin-coated pits in infected cells and reduced viral production in the presence of dynasore, ammonium chloride and bafilomycin indicate that viral entry occurs through pH-dependent endocytosis. Increase in FITC-dextran uptake (an indicator of macropinocytosis) in MAYV-infected cells, and dose-dependent infection inhibition by 5-(N-ethyl-N-isopropyl) amiloride (a macropinocytosis inhibitor), indicated that macropinocytosis is an additional entry mechanism of MAYV in vertebrate cells. Acutely infected vertebrate and invertebrate cells formed cytoplasmic or membrane-associated extracytoplasmic replication complexes. Mosquito cells showed modified hybrid cytoplasmic vesicles that supported virus replication, nucleocapsid production and maturation. Mature virus particles were released from cells by both exocytosis and budding from the cell membrane. MAYV replication was cytopathic and associated with induction of apoptosis by the intrinsic pathway, and later by the extrinsic pathway in infected vertebrate cells. Given that MAYV is expanding its geographical existence as a potential public health problem, this study lays the foundation for biological understanding that will be valuable for therapeutic and preventive interventions.

Sindbis virus is suppressed in the yellow fever mosquitoAedes aegyptiby ATG-6/Beclin-1 mediated activation of autophagy

“…Plaque assays for SINV were performed using Vero cells using standard procedures. 48 Briefly, the day before infection, 5×10 5 Vero cells were seeded on a 6-well plate to achieve a 90-100% confluent cell monolayer. 100 µl of ten-fold serial diluted virus stock/samples were inoculated into Vero cell monolayers and incubated for 1 h at 37 °C and 5% CO 2 while tilting the plate every 15 min to maintain cell hydration.…”

“…Focus-forming assays for SINV were performed using Vero cells as described previously. 48 Briefly, the day before the infection, 3×10 4 Vero cells were seeded on 96-well plates, resulting in 90-100% confluent cell monolayers. 30 µl of ten-fold serial diluted virus stock/samples were inoculated into Vero cell monolayers and incubated for 1 h in a cell culture incubator at 37 °C and 5% CO 2 .…”

Wolbachia-induced inhibition of O’nyong nyong virus inAnophelesmosquitoes is mediated by Toll signaling and modulated by cholesterol

“…RNA was in vitro transcribed from linearized NotI digested plasmid and purified using RNeasy kits (Qiagen). Two micrograms RNA was transfected into Vero cells using Lipofectamine LTX (Invitrogen) and grown as previously described (Pujhari et al 2022). Infected cells were cultured at 32ºC with 5% CO 2 for 72 hours, then supernatant of the culture was harvested, stored at -80ºC and used when required for viral infections of cells or mosquitoes.…”

“…Infected cells were cultured at 32ºC with 5% CO 2 for 72 hours, then supernatant of the culture was harvested, stored at -80ºC and used when required for viral infections of cells or mosquitoes. Virus stock titers were determined by FFU assays using Vero cells (Brault et al, 2004) as previously described (Pujhari et al 2022, Terradas et al 2022). Wolbachia (Rasgon et al, 2006) were infected with ONNV to assess for Wolbachia-mediated pathogen interference.…”

Alpha‐mannosidase‐2 modulates arbovirus infection in a pathogen‐ and Wolbachia‐specific manner in Aedes aegypti mosquitoes

“…Dengue virus (DENV) (genus Flavivirus , family Flaviviridae ) is an important human pathogen that is transmitted primarily by Aedes aegypti mosquitoes (Bhatt et al, 2013). Mayaro virus (MAYV) (genus Alphavirus , family Togaviridae ) is an emerging human pathogen that is transmitted mainly by Haemagogus janthinomys mosquitoes (Pujhari et al, 2022); however, A. aegypti mosquitoes are also competent vectors for this virus (Pereira et al, 2020). There are no approved vaccines or specific antivirals to prevent and manage disease outbreaks that are caused by either virus and thus novel strategies for disease control are needed to combat arbovirus infections.…”