A framework for assessing potential Oropouche virus-associated adverse fetal outcomes

Abstract

Several reports have suggested Oropouche virus (OROV)-related vertical transmission and adverse fetal outcomes; however, these observations require further investigation. Herein, we propose complementary experimental and epidemiological approaches to better characterize the potential role of OROV infection in fetal and neonatal disorders.

Dear Editor,

The Oropouche virus (OROV) epidemic in Brazil in 2024 attracted worldwide attention, especially due to its geographic expansion, the substantial increase in reported cases, and the first globally documented OROV-associated fatalities [1–3]. In parallel, several studies have provided evidence of vertical transmission and raised concerns about the potential impact of OROV infection on fetal development and survival, an issue that remains to be further elucidated [4–10]. Importantly, pregnancy losses among OROV-infected women were also reported in Brazil during the 1980s [11], predating current warnings about its possible role in adverse pregnancy outcomes. Herein, we propose experimental and epidemiological approaches to advance investigations into the potential role of OROV in fetal complications, aiming to support a more robust assessment of its possible impact on fetal health (Fig. 1).

Fig. 1.

Experimental and epidemiological approaches for investigating potential fetal outcomes of Oropouche virus (OROV). Experimental approaches include animal models for evaluating potential vertical transmission and fetal outcomes, as well as molecular and histopathological methods to investigate possible viral replication and damage in placental and/or fetal tissues. Epidemiological approaches include case-control studies to assess the potential association between maternal OROV infection and miscarriage and/or congenital abnormalities, as well as postnatal follow-up of exposed newborns. Evaluation of interferon (IFN) responses and interferon-stimulated genes (ISGs) may further contribute to understanding the mechanisms involved in possible OROV-associated fetal pathogenesis. NHP: non-human primate

Several case reports have described OROV RNA or antigens in placental or fetal tissues, as well as anti-OROV IgM in microcephalic newborns [4, 5, 7, 8, 10], supporting the discussion of its potential vertical transmission and effects on fetal outcomes. Despite the importance of these findings, evidence for OROV replication in fetal tissues remains limited. It is worth noting that viral infections can cause tissue damage indirectly, e.g., via immune imbalance [12]; however, demonstrating OROV replication in fetal or neonatal tissues would strengthen the biological plausibility of its role in fetal injury. In this context, strand-specific RT-PCR assays targeting positive-sense viral RNAs, including complementary RNA (cRNA) and/or viral mRNAs, could provide evidence consistent with active OROV replication. This approach could be implemented using two-step RT-PCR assays, in which reverse primers may be used to generate cDNA from positive-sense viral RNA, followed by PCR amplification of the resulting template. Furthermore, positive-sense viral RNA could be detected using signal-amplified in situ hybridization methods, such as RNAscope coupled with confocal microscopy or PrimeFlow RNA assays combined with flow cytometry. Both assays could provide complementary molecular evidence of OROV replication while enabling the identification of target cell populations in placental and/or fetal tissues, thus providing insights into possible OROV-associated fetal pathogenesis. Histopathological and immunohistochemical analyses using antibodies targeting non-structural viral proteins expressed during replication could also provide complementary evidence linking viral replication to tissue damage.

Experimental studies in animal models would also allow investigation of potential OROV-related adverse fetal outcomes. Previous reports have demonstrated vertical transmission of OROV and fetal impairment in mice, including following infection with an ancestral (historical) viral strain. However, these results were consistently observed in animals with impaired type I interferon (IFN-I) signaling [13, 14], which suggest context-dependent vertical transmission, but limit their extrapolation to natural infection. Alternatively, golden hamsters have been proposed as an experimental model for OROV pathogenesis and may represent a promising in vivo approach for investigating its possible fetal effects [15]. It would also be valuable to investigate the potential teratogenicity and other adverse fetal outcomes of OROV in animals that are likely more susceptible to viral infection, such as SJL mice [16]. Both models offer the advantage of not requiring additional genetic or immunological manipulation for viral pathogenesis research [15, 16].

Furthermore, non-human primates (NHPs) have also been used to investigate virus-associated fetal outcomes [17, 18] and could be a suitable model for OROV studies. Intrauterine infection would also provide a proof-of-concept approach to evaluate the possible fetal consequences of OROV infection in immunocompetent animals. This strategy has been used for several teratogenic viruses, including Zika virus (ZIKV) and members of the same genus as OROV (Orthobunyavirus), such as Cache Valley virus [19–22]. Intracranial inoculation in neonatal animal models and embryonated chicken eggs could also be used to evaluate potential fetal effects of OROV, as previously employed for other teratogenic orthobunyaviruses, including Aino, Akabane and Cache Valley viruses [23–25]. Importantly, these experimental studies should be conducted using both historical and recent OROV strains to evaluate possible differences in their fetal effects, and with minimally passaged viruses to avoid adaptive mutations and preserve the wild-type phenotype. These models could also be used to assess the effects of OROV at different stages of gestation.

Viral reverse genetics is also a useful tool for investigating the potential fetal outcomes of OROV, particularly regarding the influence of IFN-I on its pathogenicity, which represents a promising hypothesis for further investigation. As previously discussed, in vivo studies have demonstrated possible vertical transmission of OROV in animals without an adequate IFN-I response [13, 14]. Furthermore, IFN-I inhibition has been shown to be an important pathogenicity mechanism of orthobunyaviruses, including those with known teratogenic effects (e.g., Schmallenberg virus), and has also been implicated in the teratogenicity of Rift Valley fever virus, a virus previously classified in the same family as OROV [26, 27]. Therefore, reverse genetics could be used to manipulate the OROV NSs gene, which encodes a known IFN-I antagonist [28], to investigate its potential role in adverse fetal outcomes, as well as to identify residues, motifs, and/or domains involved in this activity. Beyond providing insights into OROV pathogenicity, these findings could inform genomic surveillance by identifying molecular signatures potentially associated with fetal injury.

In addition to laboratory analyses, well-designed epidemiological studies, such as case-control studies, could be conducted to further explore the possible association between OROV infection and fetal impairment, as previously performed for ZIKV [29]. This approach would allow estimation of the likelihood of miscarriage or developmental abnormalities in OROV-infected pregnant women, while also enabling the assessment of other factors associated with fetal damage, such as the most critical gestational period. Case-control studies could also clarify why reports of fetal outcomes from OROV have not been consistently observed over time. Prospective postnatal follow-up in case-control studies would also be timely for investigating potential later neurodevelopmental effects, including in the absence of microcephaly, as observed in ZIKV infections [30, 31]. Furthermore, pregnant women infected with OROV could undergo close immunological monitoring, particularly of IFN-I, IFN-III and interferon-stimulated genes (ISGs), such as interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), which may be modulated by OROV [32] and/or influence vertical transmission [33]. Similar analyses could also be performed in the in vivo models discussed above. Importantly, the limited number of reported OROV infections outside outbreak periods, their historical concentration in remote areas, and the sporadic and geographically restricted nature of larger outbreaks may pose challenges to case-control studies [34]. In addition, the recent decline in OROV transmission across several affected regions may further limit participant recruitment [35, 36]. These challenges could be addressed through multicenter studies and prospective surveillance strategies.

Finally, despite recent advances in our understanding of OROV, further studies are needed to determine its potential for vertical transmission and adverse fetal outcomes. Addressing these gaps requires the integration of experimental and epidemiological approaches to establish causality and elucidate the mechanisms underlying possible OROV-associated fetal complications. Such efforts are essential for improving surveillance strategies, guiding clinical management, and mitigating the potential impact of OROV infection during pregnancy.

Author contributions

LFT; NBMD: Conceptualization, Investigation, Discussion, Writing – original draft; TRRL; RFC; EFOF; LHVGG: Discussion, Writing-review & editing; JVJSJr: Conceptualization, Investigation, Discussion, Writing-review & editing, Supervision.

Funding

LHVGG received financial support from Fundação Oswaldo Cruz (FIOCRUZ) (Grant INOVA FIOCRUZ – Geração de Conhecimentos) and the National Council for Scientific and Technological Development (Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq) (Grant CNPq-444840/2023-0 and CNPq-317296/2021-3).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.