Oropouche virus causes severe congenital disease in embryonic mice

Abstract

The recent outbreak of Oropouche virus (OROV) in Latin-America with unprecedented reports of vertical transmission resulting in congenital malformations and fetal death, redefines the threat posed by this arbovirus. We discover that injection of a low inoculum of the epidemic OROV_{IRCCS-SCDC_1/2024} strain into the placenta of Swiss mice results in viral replication across embryonic organs, in particular the brain. Infected embryos present with multi-organ pathology, pneumonitis, myocarditis, steatohepatitis and severe neuropathology, including microcephaly, ventriculomegaly and neural loss. Neural cell populations are drastically altered due to massive cell death. OROV-induced neuroinflammation is marked by cytokine upregulation, microglial activation and neutrophil infiltration. When we compare to Zika virus (ZIKV) infection in the same model, a much lower inoculum of OROV than of ZIKV results in embryonic malformations, with markedly distinct neurological developmental and immune responses. Our findings establish a link between congenital OROV infection and neurodevelopmental disease, underscoring its teratogenic potential.

Using a mouse model of congenital infection, researchers show that Oropouche virus causes severe brain damage and developmental defects in the fetus, highlighting its potential to cause congenital disease in humans.

Introduction

Oropouche virus (OROV) is an arthropod-borne virus from the Orthobunyavirus genus, whose principal vector is the biting midge Culicoides paraensis¹. OROV is endemic in Central and South America¹ and has been responsible for multiple sporadic epidemic events since its first isolation and identification in 1955 in Trinidad and Tobago². Historically, OROV infections typically led to asymptomatic cases or nonspecific symptoms referred to as Oropouche fever, which is characterized by headache, arthralgia, myalgia, nausea, and vomiting, amongst others³. The small-scale Oropouche epidemics and the moderate and transient health issues caused by OROV infections led to the perception that OROV did not pose a major threat to public health. However, the 2023–2024 outbreak in Latin America marked an important turning point in light of its unprecedented magnitude, the exponential geographic spread to new countries and its impact on human health. For the first time, OROV infections were linked to fatal outcomes in infected individuals in the Americas⁴. Moreover, the Pan American Health Organization (PAHO) issued an epidemiological alert regarding possible cases of vertical transmission of OROV⁵. In 2024, five cases of OROV vertical transmission were confirmed in Brazil, with four cases of fetal death and the fifth case presenting with a congenital anomaly. Several cases, of which 22 of in utero fetal death and four cases of congenital defects, were additionally under investigation⁴. A recent study of 68 historical cases of congenital malformations identified six OROV-positive cases, including one case with severe brain defects with necrotic and apoptotic neurons, microglia and astrocytes, vacuolization and tissue atrophy. Viral RNA was detected in the brain, lung, kidney and cerebral spinal fluid, and viral antigens in brain, liver, kidney, heart and lungs tissues⁶. Altogether, the 2023–2024 OROV outbreak highlighted the threat posed by this virus to human health and to the developing fetus in particular.

Although it is recognized that vertical transmission of OROV and the associated miscarriages may result from an OROV infection, the underlying pathophysiology remains unclear. Moreover, although suspected, the link between vertical transmission and congenital disease resulting from the teratogenic potential of OROV has not been unequivocally established. Hence, there is an urgent need for a better understanding of the teratogenic potential of OROV in order to assess the potential risk of infection with this virus to the developing fetus.

Here, we examined the pathophysiology of an OROV infection after vertical transmission via intraplacental (IPL) infection in mice. We demonstrated earlier the physiological relevance of this model for the study of Zika virus (ZIKV)-induced congenital disease⁷. Given the alarming rise in OROV-associated congenital anomalies and fetal deaths, a deeper understanding of its pathogenic potential is imperative. Our study aims to shed light on the pathogenicity of OROV, which may compromise fetal development.

Results

OROV replicates efficiently in embryonic organs and causes intra-uterine growth retardation

To determine whether the epidemic OROV_{IRCCS-SCDC_1/2024}⁸ strain can affect embryonic development, we first performed intraplacental (IPL) infections at embryonic day 10.5 (E10.5) in immunocompetent outbred SWISS mice (Janvier Labs) using the same inoculum, i.e. 1000 plaque-forming units (PFU), as that used for Zika virus. However, for OROV, this inoculum resulted in 100% embryonic death and embryonic resorption. Moreover, despite a major reduction of the inoculum to only 20 PFU, the IPL at E10.5 still led to massive resorption with a harvestable rate of only 1.6%. Therefore, we performed an IPL infection in the embryonic labyrinth using 20 PFU at a later time point, i.e. E12.5 (Fig. 1a, b). Five days post-infection (d5 pi), i.e. at E17.5, the effect on embryonic development was assessed by comparing the crown-rump length and weight of the infected embryos with those of their uninfected counterparts. We found that OROV-infected embryos manifested significant intra-uterine growth retardation, as evidenced by a smaller crown-rump length (Fig. 1c, f middle) and a lower body weight (Fig. 1d) compared to mock-infected embryos at the same developmental stage, which was not due to a litter effect (Supplementary Fig. 1). OROV was found in all tested organs, such as brain, lung, heart (atrium and ventricle) and liver (Supplementary Figs. 2 and 3). When comparing the replication kinetics of OROV over the course of infection until E17.5 (d5 pi) in the different embryonic tissues (brain, lungs, heart and liver), a similar replication pattern was noted, i.e. the viral RNA load increased from E13.5 (d1 pi) to E16.5 (d4 pi; peak of replication), followed by a decrease on E17.5 (d5 pi) (Fig. 1e). However, while viral RNA levels dropped markedly in the lungs, heart and liver on E17.5, only a minor decrease was observed in the brains of embryonic mice. Moreover, notable differences were found between viral RNA levels in the embryonic brains and those in the other tissues examined on both E16.5 and E17.5. Whereas viral RNA levels were comparable in lungs, heart and liver on E16.5 (8.45 log₁₀ averaged) and E17.5 (6.51 log₁₀ averaged) levels in the brain were on average 9.67 log₁₀ and 8.99 log₁₀ on E16.5 and E17.5, respectively (Supplementary Fig. 2).

Fig. 1. Intraplacental OROV infection results in viral replication in various organs of the embryo and causes intrauterine growth retardation.

a Schematic representation of the intraplacental infection with OROV at E12.5. b Schematic representation of the experimental design of OROV infection of the fetal labyrinth 20 PFU at E12.5 and harvesting of embryos at E17.5. c Measure of the crown-rump length of mock-infected (n = 11 embryos from 2 dams) and OROV-infected (n = 21 embryos from 7 dams) embryos at E17.5. d Measure of the weight of mock-infected (n = 11 embryos from 2 dams) and OROV-infected embryos (n = 21 embryos from 7 dams) at E17.5. e Replication kinetics of OROV in the embryonic brains, lungs, hearts and liver from E13.5 to E17.5. N numbers for the viral load are shown in Supplementary Table 3. f Representative picture of a E17.5 mock-infected embryo (left), OROV-infected embryo showing IUGR (middle) and OROV-infected embryo showing hydrops (right). c, d Data are presented as mean ± standard deviation. Statistical significance of differences was determined by the unpaired two-tailed t-test with Welch’s correction (c, d). e Data are presented as mean ± S.E.M. Only statistically significant differences are shown. a Schematic representation of the intraplacental infection with OROV at E12.5 was created with BioRender.com.

OROV infection in embryonic mice induces severe multi-organ pathology

To further investigate the pathology caused by OROV in different organs (brain, spinal cord, lung, heart and liver), embryonic mice were infected via the IPL route at E12.5 and harvested at E17.5 (d5 pi). Entire embryos were subsequently processed by cryo-sectioning, stained with hematoxylin and eosin, and examined for abnormalities. The affected embryos typically exhibited delayed maturation consistent with the intrauterine growth retardation and also presented some degree of hydrops (Fig. 1f, right)). In line with the highest viral RNA load in the brain, the most severe damage was also seen in the brain, which presented severe necrotizing meningoencephalitis and hemorrhages (Fig. 2b, d). Besides the brain, the spinal cord also displayed a severe phenotype as demonstrated by the presence of hemorrhages and necrosis with mixed inflammatory infiltrations (Fig. 2f, Supplementary Figs. 4d and 5). Other organs of the embryos, including the lungs, heart and liver, were also affected. More specifically, the lungs contained mixed interstitial and focal intra-alveolar infiltrates compatible with a most prominent pneumonitis and focal pneumonia (Fig. 2h), while the heart muscle was infiltrated with a mixed inflammatory cell population indicating a moderate myocarditis (Fig. 2j). The liver showed severe hemorrhagic steatohepatitis, as proven by a positive Oil Red O staining indicating lipid accumulation (Fig. 2l, n). The immune infiltrate in the internal organs consisted mostly of mononuclear cells with extensive admixture of neutrophils (i.e. Ly6G-positive cells) (Supplementary Fig. 4b, d), especially in the necrotic areas of the CNS. Taken together, the severe lesions in the brain and spinal cord indicate neurotropism. In addition, the hydrops as well as the overall delay in organ development and the multi-organ pathology point to a strong teratogenic potential of this virus.

Fig. 2. Congenital OROV infection results in severe pathological alterations in embryonic organs.

a–n Representative hematoxylin and eosin-stained images at E17.5. Representative image of mock-infected (n = 5 from 2 dams) (a, c) and OROV-infected (n = 9 from 5 dams) (b, d) brains, orange star and green arrow indicated hemorrhages and necrosis, respectively. Representative image of mock-infected (n = 5 from 2 dams) (e) and OROV-infected (n = 9 from 5 dams) (f) spinal cord, orange star and green arrow indicated, respectively hemorrhages and inflammatory infiltration. Representative image of mock-infected (n = 5 from 2 dams) (g) and OROV-infected (n = 9 from 5 dams) (h) lungs, blue and green arrow indicated mixed interstitial and focal intra-alveolar infiltrate. Representative image of mock-infected (n = 5 from 2 dams) (i) and OROV-infected (n = 9 from 5 dams) (j) heart, green arrow indicated mixed inflammatory cell population infiltration. Representative images of mock-infected (n = 5 from 2 dams) (k, m) and OROV-infected (n = 9 from 5 dams) (l, n) liver, orange star and green arrow indicated hemorrhages and Oil Red O positive staining fat droplets. Scale bar = 100 µm.

Congenital OROV infection causes severe brain defects and extensive neural cell loss

Since OROV most severely affected fetal brains, we investigated the damage caused by this virus in more detail. To this end, we harvested embryonic brains at E17.5 (d5 pi) and performed cryo-sectioning followed by a nuclei counterstaining with DAPI, allowing visualization of the morphological alterations. Consistent with the intrauterine growth retardation, the cerebral hemispheres of OROV-infected brains were significantly reduced in size compared to the mock-infected ones at the same developmental stage (Fig. 3a–c). Normalization of head measurements to overall embryonic size demonstrated that the reduction in head size was proportional to the growth restriction in OROV-infected embryos, consistent with proportional microcephaly (Supplementary Fig. 6).

Fig. 3. Congenital OROV infection causes brain abnormalities in embryos.

Representative images of E17.5 embryonic mouse brain after mock-infection (n = 7 embryos from 2 dams) (a) and OROV-infection (b) (n = 21 embryos from 9 dams). Reduction of the brain size was assessed by measuring the brain hemisphere size (c). Ventriculomegaly was estimated by measuring the ventricle area (d). Microcephaly-like phenotypes were assessed by measuring the cortical cell density (e), the cortical length (f) and the number of DAPI-positive cells (g). h Comparative timeline of ZIKV and OROV experimental design, where ZIKV was injected at E10.5 (1000 PFU) with analysis at E18.5; OROV was injected at E12.5 (20 PFU) with analysis at E17.5. i Representative image of E17.5 OROV-infected brain hemisphere (left), cerebral cortex (middle) and striatum (right) (n = 21 embryos from 9 dams). j Representative image of E18.5 ZIKV-infected brain hemisphere (left), cerebral cortex (middle) and striatum (right) (n = 8 embryos from 4 dams). The scale bars in (a, b) represent 200 and 50 µm in the enlargements. In c–g, data are presented as mean ± standard deviation. Statistical significance of differences was determined by the unpaired two-tailed t-test with Welch’s correction (c–g). Only statistically significant differences are shown. h Comparative timeline of ZIKV and OROV experimental design was created with BioRender.com.

Although OROV-infected brains had smaller hemispheres, they showed a significant enlargement of the ventricles in comparison to mock-infected brains (Fig. 3d). Also, while the structure of the cerebral cortex in OROV-infected brains appeared similar to that in mock brains, featuring a progenitor zone (VZ-SVZ), an intermediate zone (IZ), and a cortical plate (CP) (Fig. 3a, b), significant differences were noted. OROV-infected brains displayed a microcephaly-like phenotype, characterized by a lower number of DAPI-positive cells, a reduced cell density in the cortex, as well as thinner cortices (Fig. 3e–g).

When compared to ZIKV, IPL OROV infection performed at E10.5 resulted in full embryonic death and resorption, whereas this was not the case for ZIKV, even when using a 50-fold higher inoculum. As a result, OROV IPL infections were performed at E12.5 and embryos were harvested at E17.5 (versus E18.5 for ZIKV) (Fig. 3h). Moreover, while OROV-infected brains shared common defects with ZIKV-infected brains, such as ventriculomegaly and microcephaly, the alterations in cortical layering were clearly different from those in ZIKV-infected brains, that were displaying less organized cortices than OROV-infected ones (Fig. 3i, j).

Given the neurotropic nature of OROV, we performed additional immunostainings targeting OROV and markers of different neural brain cell types to determine the viral distribution within the brain as well as which of these cells were the most affected in the OROV-infected brain. More specifically, to stain the progenitor neural cells of the cerebral cortex and striatum, we used an antibody targeting the SOX2 marker⁹. To stain the projection neurons populating the deep-layer of the cortex as well as the projection medium spiny neurons in the striatum, we used an anti-CTIP2 antibody, whereas an antibody targeting the SATB2 marker was used to stain the neurons in the upper cortical layer¹⁰.

OROV antigens were distributed throughout all layers of the cerebral cortex, with the strongest signal detected in the VZ-SVZ (Fig. 4). Co-localization of OROV antigens with SOX2 demonstrated infection of cortical progenitor cells (Fig. 4b, lower panels). Within the cortical plate, OROV infection was predominantly observed in layer V, as indicated by the presence of viral antigens in CTIP2-positive neurons (Fig. 4b, upper panels). Similarly, in the striatum, OROV antigens were mainly detected in SOX2-positive progenitor regions, although viral signal was also observed in a subset of CTIP2-positive projection medium spiny neurons (Fig. 4d).

Fig. 4. OROV viral infection in E17.5 embryonic mouse brain.

a, c Representative images of E17.5 mock-infected (n = 7 embryos from 2 dams) (a) and OROV-infected (n = 21 embryos from 7 dams) (b) cerebral cortex. b, d Representative images of E17.5 mock-infected (n = 7 embryos from 2 dams) (c) and OROV-infected (n = 21 embryos from 7 dams) (d) striatum. Light blue, green, red and white indicate DAPI, CTIP2, OROV and SOX2 staining, respectively. The scale bars represent 50 and 20 µm.

Compared to mock-infected brains, we observed a significant reduction in SOX2 and CTIP2 expression in the striatum of OROV-infected brains (Fig. 5a–d). As described previously, OROV-infected brains presented microcephaly-like phenotype with smaller cortices and fewer cells (Fig. 3e–g). The smaller cortices of OROV-infected brains had a comparable length of the ventricular-subventricular zone (VZ-SVZ) to that of the mock-infected brains (Fig. 5e, h) but a smaller intermediate zone and cortical plate (Fig. 5f–h). In addition, the cerebral cortex of OROV-infected brains showed a significant reduction in SOX2 expression (Fig. 5i, k, l) and a narrower SOX2-positive cell layer (Fig. 5j–l) compared to mock-infected brains. The percentage CTIP2-positive cells in OROV-infected brains, however, was comparable to those in mock-infected brains (Fig. 5m), albeit with a more compact distribution (Fig. 5n–p). Moreover, the upper cortical layer (II/III) had (mostly) disappeared in the majority (~75%) of the OROV-infected brains, characterized by a dramatic decrease of SATB2-positive cells (Fig. 5q) and in the length of this layer (Fig. 5r–t).

Fig. 5. Impact of OROV congenital infection on neural cell populations and brain architecture.

Measurement of number of SOX2-positive cells (a) and CTIP2-positive cells (b) in the striatum in mock-infected (n = 5 embryos from 2 dams) and OROV-infected brains (n = 8 embryos from 5 dams). Representative images of E17.5 striatum of mock-infected (c) and OROV-infected with a severe phenotype (d). Measurement of the length of the VS-SVZ zone (e), Intermediate zone (f) and the cortical plate (g) in the cerebral cortex in mock-infected (n = 5 embryos from 2 dams) and OROV-infected brains (n = 20 embryos from 7 dams). h Representative images of E17.5 of the cerebral cortex in mock-infected (left) and OROV-infected brains (right). i, m, q Measurement of number of SOX2-positive cells (i), CTIP2-positive cells (m) and SATB2-positive cells (q) in the cerebral cortex in mock-infected (n = 5 embryos from 2 dams) and OROV-infected brains (n = 8 embryos from 5 dams for SOX2 and n = 14 from 7 dams for CTIP2 and SATB2). Measurement of the length SOX2 (j), CTIP2 (n) and SATB2 (r) layers) in the cerebral cortex in mock-infected (n = 5 embryos from 2 dams) and OROV-infected brains (n = 8 embryos from 5 dams for SOX2 and n = 14 from 7 dams for CTIP2 and SATB2). Representative images of E17.5 of the cerebral cortex in mock-infected (k, o, s) and OROV-infected brains (l, p, t). Dark blue, light blue, green and red indicate DAPI, SOX2, CTIP2 and SATB2 staining, respectively. a, b, e–g, j, m, n Statistical significance of differences was determined by the unpaired two-tailed t-test with Welch’s correction. i, q Statistical significance of differences was determined by the two-tailed Mann–Whitney test. In e–g, i, j, m, n, q, r, data are presented as mean ± standard deviation. Statistically significant differences are shown; ns not significant. The scale bars represent 50 µm.

Along with the decrease in SOX2-expressing cells in the striatum and cortex, massive apoptosis in these regions was observed, as illustrated by a substantial increase in anti-cleaved caspase 3 (ACC3) expression (Fig. 6a, b). Although differences in severity were noted in OROV-infected brains, with some brains presenting a less dramatically altered phenotype, both moderately and severely affected brains displayed abundant apoptosis in the striatum, while ACC3 expression was only minor in the cerebral cortex of moderately affected brains (Fig. 6c, d). Regardless of the severity of the brain damage, the ACC3 signal was always present in the progenitor zone of the striatum and the cerebral cortex (Fig. 6c, d). In addition, the cortical defects (smaller cortical length and lower number of cells) were correlated to the number of ACC3-positive cells in the cortex (Fig. 6e, f) of OROV-infected brains (Fig. 6g–i). The decrease in SOX2- and SATB2-positive cells also correlated to the number of cortical cells undergoing apoptosis (Fig. 6j, l). Consistent with the previous observations, the number of CTIP2-positive cells did not correlate to the number of ACC3-positive cells in the cerebral cortex of OROV-infected brains (Fig. 6k).

Fig. 6. Correlation between apoptotic cell death and neuroanatomical alterations in fetal brains following congenital OROV infection.

a Percentage of anti-cleaved caspase 3 (ACC3) positive cells in the striatum in mock-infected (n = 5 embryos from 2 dams) and OROV-infected brains (n = 8 embryos from 5 dams). b Percentage of ACC3 positive cells in the cortex in mock-infected (n = 5 embryos from 2 dams) and OROV-infected brains. Representative images of a moderately affected (c) and severely affected (d) OROV-infected brains. Correlation between the number of ACC3-positive cells and the cortical length (e), the number of DAPI-positive cells (f). Representative images of mock cerebral cortex (n = 5 embryos from 2 dams) (g), and moderately (h) and severely affected (i) OROV-infected (n = 8 embryos from 5 dams) cortex. Correlation between the number of ACC3-positive cells and the number of SOX2-positive cells (j), the number of CTIP2-positive cells (k), and the number of SATB2-positive cells (l). m Percentage of the VZ-SVZ and CP area positive for ACC3 in E15.5 (n = 6 embryos from 3 dams), E16.5 (n = 6 from 2 dams) and E17.5 (n = 16 from 7 dams) OROV-infected brains. n Representative image of E15.5 OROV-infected brain hemisphere (left) and cerebral cortex (middle). o Representative image of E16.5 OROV-infected brain hemisphere (left) and cerebral cortex (middle). p Representative image of E17.5 OROV-infected brain hemisphere (left) and cerebral cortex (right). q Percentage of the VZ-SVZ and CP area positive for ACC3 signal in E18.5 ZIKV-infected (n = 8 from 4 dams) and E17.5 OROV-infected brains (n = 16 from 7 dams). r Representative image of E17.5 OROV-infected brain hemisphere (left), cerebral cortex (middle) and striatum (right). s Representative image of E18.5 ZIKV-infected brain hemisphere (left), cerebral cortex (middle) and striatum (right). Dark blue and green indicate DAPI and ACC3 staining. a, b Statistical significance of differences was determined by the unpaired two-tailed t-test with Welch’s correction. Correlation in (e, f, j–l) was determined using the two-sided Pearson r correlation test. m, q Statistical significance of differences was determined by two-way ANOVA followed by Šídák’s multiple comparisons test. a, b, e, f, j–m Data are presented as mean ± standard deviation. The scale bars represent 500 and 50 µm.

To better understand the relationship between apoptosis and the selective loss of SOX2-positive progenitors and SATB2-positive upper-layer neurons, we investigated the temporal progression of apoptosis during OROV infection. OROV-infected brains were harvested at E15.5, E16.5 and E17.5, and stained using an anti-cleaved caspase-3 (ACC3) antibody. The percentage of the VZ-SVZ and cortical plate (CP) area positive for ACC3 signal was subsequently quantified. At E15.5, both the VZ-SVZ and the CP showed a near-complete absence of cell death, with an average of 0.30 and 0.28% of the respective area staining positive for ACC3 (Fig. 6m, n). At E16.5, apoptosis had become detectable in the progenitor zone, with an average of 11% of the VZ-SVZ area positive for ACC3 staining, whereas the CP remained largely unaffected, with only 2% of its area showing an apoptotic signal (Fig. 6m, o). At E17.5, apoptosis had increased dramatically, with an average of 50% of the VZ-SVZ area found positive for ACC3 staining and some brains exhibiting up to 80% positivity (Fig. 6m, p). The CP also showed increased apoptosis at this stage, although to a lesser extent than the progenitor zone, with an average of 13.5% of the area positive testing for ACC3 (Fig. 6m, p). These findings indicate that apoptosis in OROV-infected cerebral cortex started around E16.5 and had increased massively at E17.5, with the progenitor zone representing the most severely affected region.

Finally, the pattern of distribution of the apoptotic signal observed in OROV-infected brains was specific to OROV cerebral infection, as a completely different distribution of the ACC3 signal was observed in the brains of embryos that had been injected intraplacentally with ZIKV. ZIKV-infected E18.5 brains displayed substantially less apoptosis within the progenitor zone, with ACC3-positive staining covering only approximately 3% of the VZ-SVZ area, compared to an average of 50% in OROV-infected brains (Fig. 6q–s).

In conclusion, congenital OROV infection led to a reduction in the number of SOX2-positive and CTIP2-positive cells in the striatum. The cerebral cortex also displayed a reduction in the number of SOX2-positive and SATB2-positive cells but not in CTIP2-positive cells. The decrease in progenitor cells directly correlated to a massive apoptosis, which is specific to congenital OROV infection.

A pronounced pro-inflammatory immune response in OROV-infected embryonic brains

To investigate whether and to what extent OROV had triggered an immunological response, we measured the levels of a small selection of cytokines and chemokines (IL-6, IL-1β, TNF-α, IFN-γ and CCL5) in various embryonic organs (brain, lungs, heart, and liver) collected at E17.5. All organs studied had elevated expression levels of the pro-inflammatory cytokines IL-6, IL-1β, TNF-α and IFN-γ, and the pro-inflammatory chemokine CCL5 compared to mock-infected embryonic organs (Fig. 7a). Interestingly, of all studied organs of the embryonic mice, the pro-inflammatory immune response was the strongest in the brain (Fig. 7a), which corresponded with the higher viral RNA levels in the brain than in the other organs (Fig. 1e). This finding, together with our earlier observation that OROV-infected embryonic brains were heavily infiltrated with neutrophils (Supplementary Fig. 4b), prompted us to more thoroughly investigate the neutrophil infiltrations in these brains. We therefore conducted additional immunostainings with the Ly-6G monoclonal antibody, which specifically targets the neutrophil populations. In addition, we also further characterized the microglial cells (Iba1 staining), which were earlier found to also be present in OROV-infected brains (Supplementary Fig. 4b). Whereas neutrophils were not observed in mock-infected brains, both the striatum and the cerebral cortex of OROV-infected brains showed a massive infiltration of neutrophils (Fig. 7b, c), although the number of neutrophils appeared to be higher in the striatum than in the cortex (Fig. 7d, e). The microglia in OROV-infected brains had an activated phenotype with a round shape as compared to those in mock-infected brains (Fig. 7f–h). Also, in OROV-infected brains some microglial nodules were observed, which are aggregations of activated microglial cells (Fig. 7h, right panel).

Fig. 7. Congenital OROV infection leads to an inflammatory response within the embryonic organs.

a Heatmap of expression profiles (log2 fold change) of the cytokines IL6, IL1β, TNFα, IFNγ and the chemokine CCL5 in the embryonic brain, lungs, heart and liver of OROV-infected embryos (n = 8 embryos from 5 dams) compared to mock-infected embryos at E17.5. Measurements of the number of neutrophils in the cerebral cortex (b) and striatum (c) of OROV-infected brains (n = 8 embryos from 5 dams) at E17.5. d Comparison of the number of neutrophils between the cortex and striatum of OROV-infected brains. e Representative images of E17.5 OROV-infected brains (n = 8 embryos from 5 dams) stained by immunofluorescence for neutrophils (Ly6G). f Measurement of Iba1 cells roundness in mock-infected (n = 5 embryos from 2 dams) and OROV-infected brains (n = 8 embryos from 5 dams). Representative images of microglial cells (iba1) in mock-infected (g) and OROV-infected (h) brains. Quantification of neutrophils in OROV-infected (n = 18 from 5 dams) and ZIKV-infected (n = 19 from 4 dams) cerebral cortex (ROI; width = 100 µm) (i) and striatum (j). m Quantification of Iba1 positive cells in OROV-infected (n = 18 from 7 dams) and ZIKV-infected (n = 19 from 4 dams) brain hemispheres. Representative image of OROV-infected (k) and ZIKV-infected (l) brain hemisphere (left), cerebral cortex (middle) and striatum (right), with red indicating Ly6G staining. m, n, o Representative image of OROV-infected (n) and ZIKV-infected (o) brain hemisphere with light blue indicating Iba1 staining. Statistical significance of differences was determined by unpaired two-tailed t-test with Welch’s correction (b, c, f), the two-tailed Mann–Whitney t-test (i, j, m). Statistically significant differences are shown. In b, c, f, i, j, data are presented as mean ± standard deviation. The scale bars represent 200 and 50 µm.

The immune response observed was specific to congenital OROV infection when compared to congenital ZIKV infection. ZIKV-infected brains did not present the massive infiltration of neutrophils as observed during OROV infection (Fig. 7i–l), but instead presented with a dramatic increase in microglia recruitment compared to OROV-infected brains (Fig. 7m–o). Taken together, we show that congenital OROV infection induced a pro-inflammatory immune response in all embryonic organs studied. Moreover, the inflammatory response was the strongest in embryonic brains (which also had higher viral RNA levels), as evidenced by a greater number of activated microglial cells and a pronounced recruitment of neutrophils.

Discussion

OROV circulated for decades in Central and South America. The virus was historically associated with self-limiting febrile illness or asymptomatic infections and caused only sporadic epidemic events. Consequently, OROV was previously not regarded as a significant threat to human health. The 2023–2024 outbreak in Latin America has reshaped this perception. For the first time, severe and fatal cases have been documented in young and previously healthy individuals without known comorbidities. Worryingly, recent PAHO reports describe cases of OROV vertical transmission that resulted in miscarriage. Moreover, suspected cases of OROV vertical transmission were linked to congenital disease with severe brain abnormalities reminiscent of congenital Zika syndrome (CZS), including microcephaly, ventriculomegaly and brain calcifications. Whereas vertical transmission of OROV has been confirmed clinically, proof of a causal link between in utero OROV infection and congenital disease lagged. Given the expanding geographical spread of OROV, with the emergence in previously unaffected countries [including imported cases in North America and Europe], there is an urgent need to study the consequences of OROV vertical transmission for fetal development.

Animal models have been instrumental in the investigation of viral pathogenesis. OROV pathogenesis has been studied in 3-week-old Syrian hamsters¹¹ and suckling mice¹². Wildtype (C57BL/6, CD1) adult mice are believed not to be suitable for such infection models since OROV is unable to replicate efficiently to induce disease in these mice because of the robust RIG-I-like receptor signaling pathway and the type 1 interferon (IFN) response¹³. Consequently, studies relied on IFN deficient mice to permit OROV replication and disease development. Indeed, while several studies have demonstrated that pregnant WT mice (C57/Bl6J) could sustain low levels of OROV replication, especially in the reproductive tract, with associated vertical transmission, the fetal defects were limited or absent^14,15.

Suckling mice are also not suitable to study congenital brain alterations since their cerebral cortex is already largely formed and the neurodevelopmental mechanisms after birth differ from those during embryonic development. Alternatively, to study the effect of OROV infection on embryonic brain development in WT mice, we adapted the intraplacental (IPL) ZIKV infection model that we previously developed⁷, and delivered OROV directly into the labyrinth (fetal part) of the placenta in WT SWISS mice.

In human cases of congenital OROV infection, the virus has been detected in multiple fetal organs (i.e. heart, lungs, liver, spleen and brain), with histopathology revealing widespread lesions in the infected organs and, in particular, severe brain involvement¹⁶. Consistently, IPL infection with OROV in our model led to intrauterine growth retardation, characterized by a reduced crown-rump length and a lower body weight of the embryonic mice. Robust viral replication was detected in all major organs studied (heart, lungs, liver and brain), with the highest viral RNA levels in the brain. Infected organs presented with widespread abnormalities, including hepatic steatosis and pneumonitis, with the brain and the spinal cord showing the most severe defects. Neuropathological hallmarks included hemorrhages, neural loss, and immune cells infiltration, closely mirroring findings from autopsied OROV-infected babies and fetuses^6,16. Some mouse embryos also exhibited subcutaneous edema and/or hydrops, again paralleling reported human cases.

Given the striking neurotropism of OROV, we investigated whether our model recapitulates the spectrum of congenital brain malformations reported in humans. Indeed, OROV-infected embryonic brains had smaller hemispheres than the mock-infected counterparts at the same developmental stage. Infected brains also had ventriculomegaly and a microcephaly-like phenotype (i.e. cortical thinning with reduced neural density). This indicates that OROV infection directly disrupts fetal brain development and that the brain abnormalities largely correspond to those observed in human cases. Furthermore, we demonstrated that the development of brain defects directly correlates to an in utero infection by OROV.

Because the hallmarks of congenital OROV infection included microcephaly and ventriculomegaly, we next focused on the specific impact of OROV infection on the neural populations of two major regions of the telencephalon: the cerebral cortex and the striatum, both of which are essential for higher-order brain functions (e.g. language, learning, cognition, and amongst others). The cortex is a highly organized structure consisting of six neuron layers¹⁷, whereas the striatum is a less highly defined structure compared to the cortex¹⁸. Perturbations in the development of either region are associated with lifelong neurological and psychiatric disorders. Our analyses revealed profound alterations in the neural populations of the cortex and striatum. In both the cortex and striatum of OROV-infected brains, the SOX2 population, representing the progenitor cells⁹, were markedly reduced. In addition, although the percentage of the CTIP2-positive cell population (representing the projection neurons populating the deep layer in the cortex¹⁹) appeared preserved, the distribution of these neurons was denser, reflecting impaired laminar organization. Lastly, SATB2-positive neurons from the layer II/III (representing the projection neurons in the upper cortical layer¹⁰) were drastically reduced or nearly absent. In the striatum, both SOX2-positive progenitors and CTIP2-positive spiny projection neurons²⁰ were markedly decreased, pointing to a collapse of striatal neurogenesis and circuitry. Consistent with these findings, apoptotic activity was strongly elevated in the progenitor zones of both the cerebral cortex and the striatum, hence correlating with the severe reduction in the SOX2-positive populations. The pronounced apoptotic activity observed in the progenitor zone may underlie the absence of the SATB2-positive upper cortical layer, as progenitor cells responsible for generating these neurons are undergoing cell death. This suggests that the cortical disruption could result from a direct cytotoxic effect of OROV. Together, these results show that congenital OROV infection in our model can cause devastating neural loss and structural disorganization in both the cerebral cortex and the striatum. Interestingly, loss of SOX2 progenitor cells has been linked to enlargement of the ventricles in mice²¹, thus corroborating the ventriculomegaly induced by OROV in our model. Furthermore, the compact morphology of the CTIP2 cortical layer and the almost complete absence of the upper layer are in line with the microcephalic phenotype observed in OROV-infected human fetuses.

We further assessed the immunopathological response to congenital OROV infection in our model. All infected organs displayed pro-inflammatory cytokine upregulation, with the brain showing the strongest inflammatory response, which thus appears to be linked to the higher viral load measured in the brain. OROV infection triggered a massive influx of neutrophils into the brain, which can be attributed to the higher levels of IL-6, IL-1β and TNF-α; all strong neutrophil attractants²². Moreover, neutrophil recruitment was noted in both the striatum and the cerebral cortex, although numbers tended to be higher in the striatum. This correlated with the sites of apoptosis in both regions, but with a higher prevalence in the striatum. Microglia activation and formation of microglial nodules were also evident, reflecting findings in autopsied human cases. Whereas neutrophils can have a protective role in some viral infections by contributing to viral clearance^23,24, excessive recruitment has been implicated in exacerbating neuropathology in other viral infections²⁵, raising the possibility that OROV-induced neuroinflammation contributes to fetal brain damage. The precise role of neutrophils in congenital OROV infection—whether protective, detrimental, or both—warrants further investigation.

In addition to shared neuropathological features such as ventriculomegaly and microcephaly, our comparative analysis revealed distinct cortical and immunological signatures between OROV- and ZIKV-infected brains. OROV exhibited more pronounced embryotoxicity than ZIKV, requiring a 50-fold lower inoculum to avoid complete embryonic death and resorption. However, the increased embryotoxicity also represents an important limitation when interpreting the teratogenic potential associated with congenital OROV infection. Because OROV inoculation at E10.5 resulted in near-complete embryonic lethality, infections were performed at E12.5, precluding the assessment of developmental abnormalities arising from exposure during earlier stages of neurodevelopment corresponding to the first trimester. Consequently, our results may underestimate the teratogenic impact of OROV during the first trimester of pregnancy. Importantly, however, the requirement to delay infection together with the severe pathology induced by only 20 PFU of OROV, compared with 1000 PFU of ZIKV, further underscores the remarkable embryotoxic potential of OROV. These findings raise the possibility that OROV infection during early pregnancy may more frequently result in embryonic demise or miscarriage rather than congenital disease in fetuses.

Notably, while altered cortical layering appeared more preserved in OROV-infected brains, ZIKV infection led to a markedly disorganized cortical architecture, suggesting virus-specific impacts on neurodevelopmental patterning. Furthermore, the spatial distribution of apoptotic cells, as indicated by ACC3 staining, differed substantially between the two infections, with OROV causing massive cell death in the SOX2 progenitor zone, which was not present in ZIKV-infected brains. This divergence extended to the innate immune response: congenital OROV infection was characterized by a pronounced neutrophil infiltration that was almost absent in ZIKV-infected brains, which instead exhibited extreme microglia recruitment. These findings underscore the pathogen-specific nature of OROV and ZIKV congenital infections.

While the IPL mouse model is highly valuable to investigate the physiopathology of congenital OROV infection, it also has several inherent limitations. Because the virus is injected directly into the fetal labyrinth of the placenta, this model cannot be used to study the ability of OROV to naturally cross the placental barrier during pregnancy. As a result, our study cannot determine whether the 2024 epidemic OROV strain is more efficient at crossing the placental barrier than earlier strains, a factor that might explain the first documented cases of OROV vertical transmission in humans during the 2024 epidemic. Furthermore, the model does not provide insight into the potential placental damage that OROV may induce on either the maternal or fetal side—damage that could significantly affect embryonic growth and development. For instance, the intra‑uterine growth retardation observed in some OROV vertical transmission cases may result from pathological alterations of the placenta. By injecting the virus directly into the fetal labyrinth, we artificially bias placental infection dynamics and cannot accurately assess how OROV naturally infects or affects the placenta. This limits our ability to evaluate OROV pathogenicity at the placental level. Placental OROV-induced damages could rather be studied in the WT C57/Bl6J mice that showed natural placental infection by OROV after peripheral inoculation¹⁵.

Our study design does not account for the potential role of maternal immunity nor the role of the immune system at the maternal-fetal interface on disease outcomes. Addressing crucial questions regarding vertical transmission efficiency, placental involvement, and the role of maternal immunity will therefore require additional experimental approaches and/or alternative animal models. Human placental explants could serve as a valuable and informative supplementary model for investigating placental physiopathology in response to OROV infection.

Finally, another important limitation to our study is that we did not investigate whether and how congenital OROV infection may alter the balance between direct and indirect neurogenesis, which has been shown to be a major contributor to the microcephaly phenotype caused by ZIKV²⁶. Although OROV- and ZIKV-infected embryos displayed distinct cortical phenotypes, overlapping mechanisms may contribute to the microcephaly observed following infection with either virus. Future studies will therefore be needed to dissect the respective contributions of impaired neurogenesis and the extensive progenitor cell apoptosis observed in OROV-infected brains to the development of microcephaly.

In conclusion, our findings establish the IPL mouse model as the first and highly relevant animal model to study the consequences of congenital OROV infection for the developing fetus as it recapitulates key clinical features observed in human congenital OROV cases, including multi-organ infection, severe neuropathology and hallmark developmental defects, such as microcephaly and ventriculomegaly. These findings suggest that congenital OROV infection can lead to miscarriage, neonatal death or long-term neurodevelopmental disorders, alike congenital ZIKV infection. OROV has also recently been shown to infect human trophoblasts and placental explants²⁷ and to disrupt brain organoid development²⁸. Altogether, these results firmly position OROV as an emerging public health threat, in particular for pregnant women and their developing fetuses. Enhanced surveillance of OROV circulation, systematic screening of pregnant women during outbreaks, and close clinical monitoring of pregnancies in cases of a suspected OROV infection are therefore vital to mitigate the potential impact of a congenital OROV infection.

Methods

Cells

VeroE6 cells (African green monkey kidney cells; Vero 76, clone E6; ATCC: Cat# CRL-1586) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS; HyClone) and 2 mM glutamine (Thermo Fisher Scientific). BHK-21J (baby hamster kidney fibroblasts; provided by Dr. Peter Bredenbeek at the Leiden University Medical Center (LUMC), the Netherlands) were cultured in DMEM supplemented with 10% FBS (HyClone). VeroE6 and BHK21J cells were incubated at 37 °C in presence of 5% CO₂. VeroE6 and BHK21J cells were regularly tested for mycoplasma contamination.

Oropouche virus

The Oropouche virus (OROV) strain OROV_{IRCCS-SCDC_1/2024} was isolated from a semen sample of a patient returning from Cuba and who was admitted to the IRCCS Sacro Cuore Don Calabria Hospital in Negrar di Valpolicella (Italy) in June 2024. The virus strain was kindly provided by Dr. Concetta Castilletti⁸. The whole genome sequence is available in GenBank (BankIt2843215; S segment: PP952117, M segment: PP952118, L segment: PP952119). The isolate was propagated on VeroE6 cells to generate a passage 2 of the virus stock. The infectious virus titer (expressed as plaque-forming units [PFU] per ml) was determined by plaque assay, as described previously²⁹.

Mouse experiments

Pregnant SWISS mice were purchased from Janvier Labs. Mice were housed at KU Leuven at biosafety level 3 in individually ventilated cages (type GM500, Sealsafe Plus, Tecniplast) at 21 °C, 55% humidity and 12:12 light/dark cycles. Animals were provided with food and water ad libitum, as well as with cardboard play tunnels and cotton/tissues as extra bedding material. Allocation to experimental groups was performed randomly. Housing conditions and experimental procedures were approved by the ethical committee of KU Leuven (license DMIT-190/2024 and DMIT-032/2025) following institutional guidelines approved by the Federation of European Laboratory Animal Science Associations (FELASA).

Intraplacental infection

Time-mated SWISS mice, 8–12 weeks of age, were used for the OROV and ZIKV vertical transmission experiments. All surgeries on wild-type female SWISS mice were performed at the same time, with embryonic day (E) 0.5 corresponding to the afternoon following the day of mating. At E12.5 for OROV or E10.5 for ZIKV, preoperative analgesia (0.1 mg/kg of buprenorphine; Ethiqa XR) was administered subcutaneously before induction of anesthesia. The pregnant females were anesthetized using isoflurane (Abbot Laboratories Ltd.), after which the mice were shaved. Next, a small incision (1.0–1.5 cm) was made through the lower ventral peritoneum, followed by the careful extraction of the uterine horns, which were then carefully placed on warm humidified gauze pads. The developing embryos were challenged intra-placentally with 10–20 PFU OROV, 1000 PFU ZIKV or PBS (‘mock’). After infection, incisions were sutured and disinfected. Mice were maintained on a heat pad during the whole procedure. Four to 5 h post-surgery, mice were checked for any signs of abortion.

RNA isolation and RT-qPCR

Total RNA was isolated from embryonic tissue using Trizol (Ambion, Life Technologies), according to the manufacturer’s protocol. RNA was eluted in 30 µl of RNAse free water. Quantification of OROV genome and cytokines/chemokines copy numbers was performed on a QuantStudio5 platform (Applied Biosystems) by quantitative reverse transcription PCR (RT-qPCR) using the one step RT-qPCR iTaq Universal probe kit (Ref. 1725141, Bio-Rad). After RNA isolation, each RNA isolation sample was quantified by Nanodrop and the same amount of RNA has been added to the reaction mix. Primer and probe sequences for OROV RNA and cytokine/chemokine quantification are provided in Supplementary Table 1. For OROV, Ct values were converted into a relative number of OROV RNA copies/μl using the formula y = a × ln(x) + b, where a is the slope of the standard curve, b is the y-intercept of the standard curve, and y is the Ct value for a specific sample. The standard curve for the conversion OROV RT-qPCR was defined by a 10-fold dilution series of geneblock standard with a known starting concentration. For cytokines measurement, results are expressed as Log2 fold change = 2^−∆∆Ct, where 2^−∆∆Ct = [(Ct gene of interest – Ct housekeeping gene)infected_sample – (Ct gene of interest – Ct housekeeping gene)mock_sample)]. The sequence of the cytokine primers and probes were adopted from Overbergh and colleagues^30,31.

Cryo-sectioning

Embryonic mouse heads and bodies were harvested and fixed overnight at 4 °C in 4% paraformaldehyde (PFA). Brains were dissected in 0.1 M PBS (pH 7.4). Dissected embryonic brains and bodies were cryoprotected by immersion in a sucrose gradient (first 15% sucrose in PBS followed by 30% sucrose in PBS; both overnight at 4 °C). Samples were then embedded in Polyfreeze (Sigma-Aldrich) or Epredia™ Neg-50™ Frozen Section Medium (Fisher Scientific, 12678086). Sections of 14 μm were obtained by cryo-sectioning (Leica) onto slides (Epredia SuperFrost Plus™ Adhesion slides, Fisher Scientific).

Immunofluorescence

For immunofluorescence of brain and body sections, antigen retrieval was performed at 95 °C for 15 min before incubation with primary antibodies. Slides were then incubated in blocking solution (Normal Donkey Serum 10%, Bioconnect Life Sciences) for 1 h at room temperature. Sections were incubated with primary antibodies (Supplementary Table 2) overnight at 4 °C. The following day, slides were stained using the secondary antibodies (Supplementary Table 2) and nuclei were counterstained with DAPI (1:1000) for 2 h at room temperature. Slides were mounted in Dako Fluorescence Mounting Medium (Agilent).

RNAScope

Probe V-OROV-S-O5-sense-C1 (Advanced Cell Diagnostics, Ref. 1811921-C1) was custom-designed against the S segment nucleoprotein gene, reverse-complement of nt 10-937 of the GenBank PP952117.3 sequence³². This probe is publicly available. Manual RNAscope assays were performed following the manufacturer protocol using the Multiplex Fluorescent Detection Kit v2 (Advanced Cell Diagnostics, Ref. 323110) on 14 µm E17.5 whole embryo cryosections. Signal amplification was followed by development with the fluorophore dye Opal 650 (Akoya Biosciences, Cat n° FP1496001KT) and nuclei were counterstained with DAPI (1:1000) for 20 min. Slides were mounted in Dako Fluorescence Mounting Medium (Agilent).

Hematoxylin and eosin staining

Brains and whole embryonic body sections were stained for hematoxylin and eosin using the H&E Staining Kit (Hematoxylin and Eosin, Abcam, ab245880) following the manufacturer’s protocol.

Confocal microscopy and analysis

Confocal images of sections of embryonic mouse brains and whole bodies were acquired using a Leica DMi8 confocal spinning disc microscope at a ×25 magnification. Image analysis and processing were performed with ImageJ 1.42q 276 (Wayne Rasband, National Institutes of Health), Fiji (v2.0.0-rc-54/ 1.51h; https://imagej.net/Fiji) or Cellpose³³ software packages.

Statistics

Statistical analyses were performed using GraphPad Prism v9.3.1. Results were first tested for normality using the Shapiro–Wilk test. In case of a normal distribution of the data, an unpaired t-test with Welch’s correction was performed. When the data did not follow a normal distribution, a Mann–Whitney test was performed. Differences were considered statistically significant when p < 0.05.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Author contributions

M.D.: conceptualization, planning, coordination and execution of the work. R.M.V.: histopathology pictures acquisition and interpretation. N.C., M.F., and S.H.: technical assistance. A.H.: image analysis and analysis software assistance. C.C.: access to OROV-IRCCS strain. S.J.F.K. and J.N.: securing of funding, conceptualization. S.J.F.K. and M.D. wrote the manuscript with contributions from J.N., and comments from all authors.

Peer review

Peer review information

Nature Communications thanks Ashley St. John and the other anonymous reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Funding

This work was funded by the DURABLE project (to J.N.). The DURABLE project has been co-funded by the European Union, under the EU4Health Programme (EU4H), project no. 101102733. Views and opinions expressed are, however, those of the author(s) only and do not necessarily reflect those of the European Union. Neither the European Union nor the granting authority can be held responsible for them. This project has received funding from the European Union’s Horizon Europe research and innovation program under Grant Agreement no. 101137229. This work was also supported by the Belgian Federal Government for the VirusBank Platform. The funders had no role in the study design, data collection and interpretation, or the decision to submit the work for publication.

Data availability

All data generated and analyzed in this paper are included in this article. The raw immunostaining and hematoxylin-eosin (H&E) image datasets supporting the findings of this study are not publicly deposited because of their size and the lack of a dedicated repository for these data; however, they are available from the corresponding author upon reasonable request. Supplementary Figs. are provided with this paper. Source data are provided with this paper.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at https://doi.org/10.1038/s41467-026-77294-6.