key: cord-0268856-airbjqie
authors: Patel, Aum R.; Dulcey, Melissa; Abid, Nabil; Cash, Melanie N.; Dailey, Jordan; Salemi, Marco; Mavian, Carla; Vittor, Amy Y.
title: Infectivity of three Mayaro Virus (Genus Alphavirus, Family Togaviridae) geographic isolates in human cell lines
date: 2021-09-29
journal: bioRxiv
DOI: 10.1101/2021.09.29.462323
sha: 558f1b5486ea1c8e761aec596790a76e6ca9d046
doc_id: 268856
cord_uid: airbjqie

Mayaro virus (MAYV) is an emergent arthropod-borne virus that causes an acute febrile illness accompanied by arthralgia, similar to chikungunya virus. Increasing urbanization of MAYV outbreaks in the Americas has led to concerns that this virus could further expand its geographic range. Given the potential importance of this pathogen, we sought to fill some critical gaps in knowledge regarding MAYV infectivity and geographic variation. This study describes the cytopathogenicity of MAYV in human dermal fibroblasts, human skeletal muscle satellite cells, human embryonic kidney cells (HEK), peripherally derived human macrophages, and Vero cells. MAYV strain isolated from Bolivia (MAYV-U) infected cells more rapidly compared to MAYV strains isolated in Peru and Brazil (MAYV-P; MAYV-B), with high titers (1×108 pfu/ml) peaking at 37 hours post infection. MAYV-U also caused the most cytopathic effect in a time dependent manner. Furthermore, differently from the other two prototypic strains, MAYV-U harbors unique mutations in the E2 protein, D60G and S205F, likely to interact with the host cell receptor, which may explain the observed differences in infectivity. We further demonstrate that pre-treatment of cells with interferon-β inhibited viral replication in a dose-dependent manner. Together, these findings advance our understanding of MAYV infection of human target cells and provide initial data regarding MAYV phenotypic variation according to geography. Author Summary Arthropod-borne viruses are of great public health concern, causing epidemics worldwide due to climate change, changes in land use, rapid urbanization, and the expanding geographic ranges of suitable vectors. Among these viruses, Mayaro is an emerging virus for which little is currently known. This study aims to answer fundamental questions of Mayaro virus biology using three geographically distinct viral strains to examine variability in infection kinetics and infectivity in susceptible cell types. We found one geographic isolate to have accelerated infection kinetics and increased cell damage because of infection. To better understand what was unique about this isolate, we compared their envelope protein, which is critical for entry into a cell. We found that the isolate with increased replication kinetics possessed mutations at sites that may promote viral entry, which could explain these findings. Together, these findings further our understanding of Mayaro virus biology and provide insight into factors that contribute to Mayaro transmission and infectivity.

Arthropod-borne viruses are of great public health concern, causing epidemics worldwide due to climate 34 change, changes in land use, rapid urbanization, and the expanding geographic ranges of suitable 35 vectors. Among these viruses, Mayaro is an emerging virus for which little is currently known. This study 36 aims to answer fundamental questions of Mayaro virus biology using three geographically distinct viral 37 strains to examine variability in infection kinetics and infectivity in susceptible cell types. We found one 38 geographic isolate to have accelerated infection kinetics and increased cell damage because of infection.

To better understand what was unique about this isolate, we compared their envelope protein, which is 40 critical for entry into a cell. We found that the isolate with increased replication kinetics possessed 41 mutations at sites that may promote viral entry, which could explain these findings. Together, these DMEM was added to each well to prevent the cells from drying out. The plates were incubated at 37°C, 122 5% CO2 for 1 hr to allow for viral adsorption before the media was aspirated and replaced with 0.5 ml of 123 1% methyl-cellulose medium (5% EqualFETAL™, 2% Pen Strep, 2% L-Glut in 2x E-MEM). Cells were 124 incubated for 3 days before the media was removed and stained with 0.25% crystal violet (30% methanol; 125 70% dH2O) for 30 minutes. The crystal violet was then aspirated and the plates were washed with water.

Plaques were counted manually and the concentration in plaque-forming units/ml (pfu/ml) was calculated. was then added for a 3-hr adsorption at 37°C and 5% CO2. Following adsorption, cells were rewashed 170 once with DPBS, fresh media was added, and then incubated for 21 hours at 37°C and 5% CO2. After 21 171 hours, supernatants were harvested, centrifuged at 1200 xg for 10 minutes and frozen at -80°C (18).

To assess viral replication in cells treated with IFN after infection, cells were infected first with MAYV-U at 173 a MOI of 1 for a 4-hr adsorption at 37°C and 5% CO2. Cells were then washed with DPBS and fresh 174 media containing IFN-β or IFN-γ was added at the aforementioned concentrations in duplicate for a 21-hr 175 incubation. After 21 hrs, supernatants were harvested, centrifuged at 1200 xg for 10 minutes and frozen 176 at -80°C (18). All supernatants were titered following the plaque assay protocol described earlier. (Fig 1A) , suggesting the cells were quickly infected. HEK cells 237 also became infected within 24 hrs with some loss in cell density with increasing time, suggestive of 238 MAYV induced cytopathic effects (Fig 1B) . SkMc cells were readily infected based on detectable 239 envelope at 24 hpi ( Fig 1C) . Viral envelope was not completely detectable on Vero cells at 24 hpi 240 compared to other cell lines but was present on nearly all cells by 48 hpi, and it drastically reduced at 72 241 hpi due to substantial cell death as shown by the lack of DAPI positive cells (Fig 1D) . While some 242 macrophages became infected with MAYV within the first 24 hpi, infection was only minimally detectable 243 thereafter (Fig 1E) . In fibroblasts and SkMc, as time progressed, we observed condensation and 244 fragmentation of the nuclei, known as karyorrhexis and pyknosis, respectively, which suggested that the isolates, Vero cells, which have no natural innate antiviral mechanisms, showed the greatest loss in cell 254 viability (Fig 2A) . HEK293T cells, however, showed little CPE across viral isolates (Fig 2B) . (28, 29) . Based on the structural modeling, we conclude that 299 the unique mutations found in MAYV-U, that map to the exposed region of the E2 glycoprotein facing 300 cellular surface, are likely to alter the interaction of the virus with its host receptor and therefore may be 301 related to differential fitness. 

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We would like to thank Aya Tal