Genomic characterization of Sabiá virus in Brazil, 2019-2020: Implications for diagnostics, virus evolution, and receptor binding

Abstract

Between December 2019 and January 2020, two patients suspected of having severe yellow fever were admitted to a tertiary healthcare facility in São Paulo, Brazil, presenting with acute hemorrhagic syndrome and neurological alterations; both cases had fatal outcomes. Upon admission, both tested negative for yellow fever viral RNA, and Sabiá virus (SABV), a New World arenavirus, was identified as the causative pathogen. To date, only four humans naturally acquired SABV infections have been confirmed, all fatal and linked to rural settings. We applied next-generation sequencing to generate complete and near-complete genomes from two patients (SP17 and SP19). Existing molecular diagnostics failed to detect SABV; therefore, new molecular tests were developed. Genetic analyses of SP17 and SP19 genomes along with other arenaviruses, revealed that the new cases were genetically diverse, showing 93-98.2% amino acid identity at the NP level among SP17, SP19, and the 1990 reference strain (SPH114202). Time-scaled phylogenetic analyses confirmed that SP17 and SP19 were not epidemiologically linked and suggested that SABV has been circulating undetected in Brazil for over a century. Additionally, homology modeling and structure-based mapping provided insights into SABV receptor-binding sequence conservation, suggesting that SABV shares similar receptor binding structure to other clade B arenaviruses, despite some amino acid variation around receptor binding site. Our findings underscore the need for retrospective and prospective surveillance of undiagnosed hemorrhagic fever cases to assess the public health impact of SABV in Brazil.

Fig 1. Overview of reported SABV and other human pathogenic arenavirus infections in the Americas.

(A) Distribution of human pathogenic NWA, including species, year of first detection, state, country, and host information. Circles correspond to reported outbreaks (with case numbers) and are coloured according to NWA clade. São Paulo state, Brazil, is highlighted in dark grey on the map. (B) Map of municipalities of São Paulo state, Brazil, with coloured municipalities indicating locations where naturally occurring SABV infections were detected. Distances between cases are: 155 km between Case 1 (1st reported SABV case) and Case 2, 380 km from Case 2 to SP19, and 315 km between SP19 and SP17. Timeline summarizes events related to naturally occurring and laboratory-acquired SABV infections, with SP19 and SP17 highlighted in red. (C) and (D) ML phylogenetic trees of arenavirus four major clades for L segment and S segment, respectively (Dataset A, https://github.com/CADDE-CENTRE/SABV_Brazil). Tips include virus strain names and countries of identification for clade B NWA viral species. Scale bar = nucleotide substitutions per site (s/s), H = hemorrhagic, L = large, S = small, bp = base pairs, BR = Brazil, AR = Argentina, BO = Bolivia, VE = Venezuela. Maps in panels A and B were created in R version 4.0.1 using the ggplot2, sf, rnaturalearth, geobr, tmap, lwgeom, units, and mapview packages. Basemap data: Natural Earth (http://www.naturalearthdata.com, public domain) and geobr (based on official IBGE shapefiles, public domain).

Fig 2. SABV genome structure and primer-binding site mismatches.

(A) SABV genome structure, featuring an enveloped RNA with a single-stranded ambisense configuration. Segment L encodes the Z matrix protein and the L polymerase protein, while Segment S is responsible for encoding the GP and NP, separated by non-coding intergenic regions (IGR). Linear genomes are flanked by conserved 5’ and 3’ untranslated regions (UTRs) crucial for viral replication and translation processes [13]. (B) The upper section displays new strain sequences relative to the SABV reference, primer locations (Bowen et al. [12]), and variant positions in black. The lower part presents primer and SABV sequences at binding sites, with colours indicating primer mismatches and pale grey bars indicating inosine sites. Note: primers are shown in their published 5′ → 3′ orientation. Reverse primers, such as 1010C (5′–TCIGGIGAIGGITGGCC–3′), anneal to the genome by reverse complementarity and therefore may not appear as direct sequence matches when aligned against the 5′–3′ representation of the negative-sense genome. Vs = versus, Z = matrix protein Z, L = RNA-dependent RNA polymerase protein L, GPC = glycoprotein precursor, NP = nucleocapsid protein, IGR = non-coding intergenic regions, UTR = untranslated regions.

Fig 3. Time-calibrated phylogenies and estimated divergence times for SABV and other arenaviruses.

We employ sequence data from SP17 with higher genome coverage for analysis. M = millions, Mya = million years. Inferences were performed for both S (A) and L (B) segments. (C) Posterior distributions for the dates of segments S and L most recent common ancestor.

Fig 4. Structural analysis of SABV GP1 mutations.

To facilitate comparative analysis of amino acid conservation between the SABV GP1 reference and contemporaneous strains we generated a homology model of SABV GP1 (SP17 strain) using AlphaFold3 [17]. (A) Mapping the TfR1-interaction region, an important determinant of zoonotic potential [18], on the surface of the GP1. (Left) Structure of MACV GP1 (PDB no. 3KAS) is shown as a rainbow-colored cartoon from blue (N-) to red (C-terminus) with N-linked glycosylation sites displayed as grey spheres. (Right) Structure of MACV GP1 is shown in white-colored surface representation, with hTfR1-binding residues colored red (determined with PDBePISA [19]. A GP1-interacting chain of hTfR1 is shown as an orange-colored ribbon. The side chain of hTfR1-Tyr211 is shown as a stick. (B) Mapping amino acid conservation between SABV GP1 from the reference and contemporaneous strains. (Left) AlphaFold model of the new SABV strain GP1 (SP17), with putative N-linked glycosylation sites displayed as grey spheres. For fair comparison, only residues 80–234 are shown. Substitutions N88S, N208D, and R216T are displayed as sticks. (Right) The model is shown in surface representation and colored white. Amino acid residues dissimilar to the reference SABV GP1 are colored blue. A GP1-interacting chain of hTfR1 (PDB no. 3KAS) is overlaid onto the homology model of SABV GP1 to indicate region of the putative TfR1 binding site. [20].