Oropouche virus as an emerging cause of acute febrile illness in Colombia

Abstract

Arbovirus infections are frequent causes of acute febrile illness (AFI) in tropical countries. We conducted health facility-based AFI surveillance at four sites in Colombia (Cucuta, Cali, Villavicencio, Leticia) during 2019-2022. Demographic, clinical and risk factor data were collected from persons with AFI that consented to participate in the study (n = 2,967). Serologic specimens were obtained and tested for multiple pathogens by RT-PCR and rapid test (Antigen/IgM), with 20.7% identified as dengue positive from combined testing. Oropouche virus (OROV) was initially detected in serum by metagenomic next-generation sequencing (mNGS) and virus target capture in a patient from Cúcuta. Three additional infections from Leticia were confirmed by conventional PCR, sequenced, and isolated in tissue culture. Phylogenetic analysis determined there have been at least two independent OROV introductions into Colombia. To assess OROV spread, a RT-qPCR dual-target assay was developed which identified 87/791 (10.9%) viremic cases in AFI specimens from Cali (3/53), Cucuta (3/19), Villavicencio (38/566), and Leticia (43/153). In parallel, an automated anti-nucleocapsid antibody assay detected IgM in 27/503 (5.4%) and IgG in 92/568 (16.2%) patients screened, for which 24/68 (35.3%) of PCR positives had antibodies. Dengue was found primarily in people aged <18 years and linked to several clinical manifestations (weakness, skin rash and petechiae), whereas Oropouche cases were associated with the location, climate phase, and odynophagia symptom. Our results confirm OROV as an emerging pathogen and recommend increased surveillance to determine its burden as a cause of AFI in Colombia.

Keywords: Colombia; NGS; Oropouche; RT–PCR; acute febrile illness; bunyavirus; fever; serology.

Graphical abstract

Figure 1.

AFI surveillance and detection of Oropouche. A. Map of Colombia showing the four AFI surveillance sites and the city of Medellín where the testing laboratory is located. B. AFI pre-screening results by site, indicating the number of positive Dengue cases and those without a diagnosis. C. mNGS (orange) and CVRP (blue) coverage plots for L, M, and S genome segments of patient 0200178W. D. Oropouche virus was isolated on Vero cells by infecting sera from three individuals.

Figure 2.

Independent introductions and cryptic circulation of Oropouche in Colombia cluster with Ecuadorian strains and all descend from Peru. ML phylogenetic trees of were reconstructed with three strains sequenced here (red) and all available Oropouche references in GenBank. Genetic distances are measured in substitutions/site/year for the A. S segment, B. M segment, and C. L segment. All nodes showed support value indicated by bootstrap >55. In all cases the tips were coded to represent the geographic location denoted in the legend. Curved-red arrows highlight the local evolutionary link between the initial case (FCT00025) reported in Turbaco and the 0200178W index from Cucuta. The straight red arrows represent the segregation of strains from Leticia which occurred before 2017 as an independent introduction directly from Peru.

Figure 3.

Numerous cases of Oropouche fever detected in Colombia. A. Genomic depiction of where primers/probes are situated on L and M segments for RT-qPCR. The nucleocapsid protein encoded on the S segment was used for serology. B. RT-qPCR results, plotting the number of viremic OROV cases versus cycle threshold (Ct). C. Anti-OROV nucleocapsid IgG (left) and IgM (right) antibodies detected Colombians with AFI. Greyzone (0.8-1.0) and S/CO ≥ 1.0 values were considered positive. D. (left) Western blot detection of OROV-infected Vero lysate using patient plasma (IgG S/CO values are indicated). The band at 25 kDa is nucleocapsid. (right) Competitive inhibition assay showing loss of anti-nucleocapsid reactivity in presence of excess recombinant protein (lanes 2, 4). E. Summary depiction of RT–PCR and antibody positives.

Figure 4.

OROV cases are present throughout Colombia but concentrated near the Amazon River basin. A. OROV molecular results by year and month for each site. Stacked histograms indicate the number of RT-qPCR positives (red), RT-qPCR negatives (grey), and remaining AFI samples not tested (blue). B. OROV serology results by year and month for each site. Stacked histograms indicate the number of IgM positives (green), IgG positives (red), dual IgM/IgG positives (yellow), antibody negatives (grey), and remaining AFI samples not tested (blue).