Comparative analysis of host responses to Oropouche virus infections in placental cell infection models

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2022-2024 saw a major outbreak of midge-borne Oropouche virus (OROV, Peribunyaviridae) that affected Brazil and countries across the region and was associated with a novel reassortant of this pathogen. The description of fetal deaths, microcephaly and congenital malformations in newborns led to the question whether such pathologies had been previously been overlooked or are associated with novel properties of reassortant OROV, raising the question of infection and transfer across the placenta. Here we compared infection of cultured placental cells with an earlier OROV isolate and a Cuban isolate from 2024. Our data suggest minor but noticeable differences between these OROV and respective host cell responses, for example relating to IFNL3. It remains to be investigated whether characteristics of these different isolates translate to effects on fetal development, but these findings suggest that cellular responses can differ between older and recent isolates of this virus.For transcriptome analysis, BeWo and JEG-3 cells (5x105 cells per well of a 6-well plate) were infected with OROV1_2010 or OROV2_2024 at a MOI of 0.1 or 1.0 for 1 hour at 37°C with 5% CO2. After incubation, virus media was removed and fresh respective cell media with 10% FBS and 1% P/S were added. After 18 hours, cell culture supernatant was removed, and cells were lysed using TRIzol (Invitrogen), according to the manufacturer’s instructions, and lysate was stored at -80°C. TRIzol lysates were sent to Genewiz (Azenta Life Sciences) for RNA purification, preparation of sequencing libraries and sequencing. mRNAs were enriched using Oligo(dT) beads, and RNA sequencing libraries were prepared using a NEBNext Ultra II RNA Library Kit for Illumina (NEB). The sequencing libraries were then validated on an Agilent Fragment Analyzer (Agilent Tchnologies) followed by quantification on a Qubit Fluorometer (Invitrogen). Libraries were sequenced to generate 150-bp paired-end reads using an Illumina sequencing platform. The raw sequencing data (bcl files) generated were converted into fastq files and de-multiplexed with Illumina bcl2fastq software. Sequences were then aligned to the human reference genome (hg38) with STAR (version 2.6.8a) [29]. Differential gene expression was performed using DESeq2 (v1.30.1) [30] in R (v3.8.3). Differentially expressed genes (DEGs) were defined as cellular genes with log2 (fold-change) >1 and adjusted P value <0.05. Gene Ontology (GO) analysis was carried out with DAVID (National Institutes of Health, USA) [31]. Sample meta data is deposited here.

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