Emergence of Oropouche fever in Latin America: a narrative review

Abstract

Since its discovery in 1955, the incidence and geographical spread of reported Oropouche virus (OROV) infections have increased. Oropouche fever has been suggested to be one of the most important vector-borne diseases in Latin America. However, both literature on OROV and genomic sequence availability are scarce, with few contributing laboratories worldwide. Three reassortant OROV glycoprotein gene variants termed Iquitos, Madre de Dios, and Perdões virus have been described from humans and non-human primates. OROV predominantly causes acute febrile illness, but severe neurological disease such as meningoencephalitis can occur. Due to unspecific symptoms, laboratory diagnostics are crucial. Several laboratory tests have been developed but robust commercial tests are hardly available. Although OROV is mainly transmitted by biting midges, it has also been detected in several mosquito species and a wide range of vertebrate hosts, which likely facilitates its widespread emergence. However, potential non-human vertebrate reservoirs have not been systematically studied. Robust animal models to investigate pathogenesis and immune responses are not available. Epidemiology, pathogenesis, transmission cycle, cross-protection from infections with OROV reassortants, and the natural history of infection remain unclear. This Review identifies Oropouche fever as a neglected disease and offers recommendations to address existing knowledge gaps, enable risk assessments, and ensure effective public health responses.

Full text

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Emergence of Oropouche fever in Latin America: a narrative review
THE LANCET Infectious Diseases | Review |
Konrad
M
Wesselmann
1
,
Ignacio
Postigo-Hidalgo
2
,
Laura
Pezzi
1,3
,
Edmilson
F
de
Oliveira-Filho
2
,
Carlo
Fischer
2
,
Xavier
de
Lamballerie
1,3
,
Jan Felix Drexler
2
1Unité des Virus Émergents (UVE: Aix-Marseille Univ-IRD 190-Inserm 1207), Marseille, France
2Institute of Virology, Charité- Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany
3Centre National de Référence (CNR) des Arbovirus, Marseille, France (L Pezzi, Prof X de Lamballerie); German Centre for Infection Research (DZIF), Berlin, Germany
https://doi.org/10.1016/S1473-3099(23)00740-5
Since its discovery in 1955, the incidence and geographical spread of reported Oropouche virus (OROV) infections have increased. Oropouche
fever has been suggested to be one of the most important vector-borne diseases in Latin America. However, both literature on OROV and
genomic sequence availability are scarce, with few contributing laboratories worldwide. Three reassortant OROV glycoprotein gene variants
termed Iquitos, Madre de Dios, and Perdões virus have been described from humans and non-human primates. OROV predominantly causes
acute febrile illness, but severe neurological disease such as meningoencephalitis can occur. Due to unspecific symptoms, laboratory
diagnostics are crucial. Several laboratory tests have been developed but robust commercial tests are hardly available. Although OROV is
mainly transmitted by biting midges, it has also been detected in several mosquito species and a wide range of vertebrate hosts, which likely
facilitates its widespread emergence. However, potential non-human vertebrate reservoirs have not been systematically studied. Robust animal
models to investigate pathogenesis and immune responses are not available. Epidemiology, pathogenesis, transmission cycle, cross- protection
from infections with OROV reassortants, and the natural history of infection remain unclear. This Review identifies Oropouche fever as a
neglected disease and offers recommendations to address existing knowledge gaps, enable risk assessments, and ensure effective public health
responses.
Key messages
 Over the past 70 years, a notable increase in the incidence and geographical spread of reported Oropouche virus (OROV) infections
has been observed, highlighting a growing public health concern.
 The OROV genome is tri-segmented, which allows for reassortment. Three OROV reassortants are known, among which Madre de
Dios and Iquitos viruses have been associated with human diseases.
 Human OROV infection occurs through the bites of midges, but its presence in various mosquito species and a wide range of
vertebrate hosts contributes to the potential for widespread emergence of OROV. However, the virus’ transmission cycle is poorly
understood.
 OROV causes febrile symptoms indistinguishable from common pathogens endemic to the Americas. Occasionally OROV infection
can result in severe conditions, such as meningoencephalitis.
 Due to the virus’ unspecific symptoms, laboratory diagnostics are crucial. Several in-house molecular and serological tests have
been developed, but robust commercial tests are scarcely available.
 Animal models of OROV infection are restricted to rodent models. The pathogenesis of OROV and its reassortants is poorly
understood. The possibility and extent of repeated infection by different OROV glycoprotein reassortants are unknown.
 The gaps in understanding the epidemiology, ecology, and pathogenesis of OROV, including its reassortants, are substantial.
Addressing these knowledge gaps is crucial for developing risk assessments and effective public health strategies to combat
Oropouche fever, a prototypical neglected disease.

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Introduction
Vector-borne diseases cause up to 28·8% of all emerging infectious
diseases.1 Arthropod-borne viruses (ie, arboviruses) comprise 39% of
all pathogenic viruses discovered in humans between 1897 and 2010,
and are a main cause of vector-borne diseases.2,3 The importance of
arboviruses is best illustrated by the dengue virus (DENV), which is
the most important arbovirus infecting over 100 000 000 humans
annually.4 In contrast to vector- borne parasites, the global burden of
arboviruses increased between 2007 and 2017.3 The true burden of
arboviral disease is probably still underestimated.5,6 Poor knowledge of
transmission cycles, non-specific clinical presentations, and scarcity of
robust diagnostic tools are among the main problems that make
arboviral infections and their effect on patient and public health
difficult to identify.7
Latin America is considered a hot spot of emerging arboviruses due to
its large biodiversity including relevant vertebrate reservoirs such as
bats, rodents, and primates, and multiple invertebrate taxa.8,9
Moreover, key emergence factors are present in the region, comprising
(1) ecological and socioeconomic factors, especially rapid population
growth and often arbitrary urbanisation; (2) favourable climatic
conditions for vector establishment and proliferation, and large
diversity of ecosystems; and (3) intense human migration and major
changes in land use, such as deforestation, illegal mining, and
intensification of agriculture.5,10,11 Along with increased global
connectivity, the Americas have therefore seen the emergence (and re-
emergence) of several arboviruses, such as DENV, chikungunya virus
(CHIKV), West Nile virus, and Zika virus (ZIKV), during the past 50
years. Nonetheless, only DENV and CHIKV are officially listed as
pathogens causing a neglected tropical disease (NTD) by WHO.12
However, the Pan American Health Organization has begun to
acknowledge additional arboviruses as emergent and has started
issuing guidelines for their diagnosis, treatment, and laboratory
detection in the region.13,14
The Pan American Health Organization considers DENV to be the
biggest threat in the Americas because of its four serotypes and its well
established urban transmission cycle.15 In addition, since their
emergence on the American continent in 2013, CHIKV and ZIKV have
caused millions of infections in the region, incurring approximately
140 000 disability-adjusted life- years every year and, in the case of
ZIKV, congenital mal- formations.16–20 Other arboviruses, such as
Oropouche virus (OROV), Mayaro virus,21 or Venezuelan equine
encephalitis virus,22,23 are also considered emergent in the region.
Before the recent emergence of CHIKV and ZIKV from 2013 onwards,
OROV was claimed to be the second most frequent arbovirus in Brazil
after DENV, allegedly accounting for more than half a million
estimated cases in Central and South America since its first
identification.24,25
Upon closer examination of OROV cases, estimates of its relevance
have relied on a few small-scale studies;26–28 therefore, the true burden
of OROV-induced disease remains unknown. Although Oropouche
fever could be a prototypic NTD, incurring considerable health burden
in affected countries, knowledge of OROV and the disease it causes is
scarce.
This Review discusses the current state of research on OROV and
identifies research gaps requiring further investigation to enhance
patient care, enable risk assessments, and ensure effective public health
responses to Oropouche fever.
Genetic features of OROV and its reassortants
OROV’s genomic sequence availability
OROV belongs to the Simbu serogroup of the viral genus
Orthobunyavirus in the Peribunyaviridae family. The OROV genome
consists of three single-stranded, negative-sense RNA molecules:
small (S), medium (M), and large (L) segment. The S segment encodes
the nucleocapsid and a non-structural protein (NSs) in overlapping
open reading frames; the M segment encodes the two glycoproteins Gc
and Gn, and a non- structural protein (NSm); and the L segment
encodes the RNA-dependent RNA polymerase (figure 1A).29–31
513 sequences, including 50 sequences of OROV reassortants, were
retrieved from GenBank. Most of those sequences corresponded to the
S segment (n=256); this could be because this segment is the most
conserved and the target of most PCR systems (compared with 142 M-
derived sequences and 115 L-derived sequences), which restricts our
knowledge and analysis of those genomic segments (figure 1B). This
small number of sequences probably represents only a fraction of the
existing OROV genetic diversity that is additionally biased towards
few sampling sites and dates.
OROV reassortants
OROV’s tri-segmented genome is susceptible to reassortment when
two viruses co-infect a single cell, resulting in progeny with mixed
genomic segments.30–32 These events are important drivers of genetic
divergence, potentially altering vector competence and disease severity
and therefore facilitating viral emergence.31,32 Among
orthobunyaviruses circulating in South America, such as Fort Sherman
virus and Jatobal virus, reassortment events are frequent.30,33 Because
arboviral surveillance in the Americas is scarce, the number of OROV
reassortants is possibly underestimated.30,34 Furthermore, experimental
evidence shows possible reassortment between OROV and non-
American ortho- bunyaviruses, such as Schmallenberg, Bunyamwera,
and Oya viruses,32 underscoring the risk of new OROV reassortants
emerging in the future.
Three OROV reassortants termed Iquitos virus (IQTV), Madre de Dios
virus (MDDV), and Perdões virus (PDEV) have been identified. These
reassortants contain the S and L segments of OROV, the M segment of
unknown viruses, and, in the case of IQTV, an MDDV-related M
segment, as highlighted by the high sequence distances of the
reassortant M segments as compared with OROV (figure 2A) and the
common ancestor of MDDV and IQTV in an M-based pyhlogenetic
tree (figure 2B).29,34,37,38,39 The M segment’s genetic diversity could be
influenced by evolutionary pressure on the glycoproteins targeted by
antibody-mediated immune response29 and suggests that M segment-
based reassortment could provide evolutionary benefits for evading
population-level immunity, whereas S and L segments could be
functionally linked due to the interaction of derived proteins during
replication.29,40
Data concerning the serological relationship between OROV and its
reassortants, including IQTV, are scarce. Experiments have shown that
mouse antisera against IQTV only weakly neutralise OROV, indicating
distinct serotypes, with no reciprocal effect observed.38 IQTV infection
in two Peruvian patients triggered an increase in potentially pre-
existing OROV immune responses, suggesting conserved epitopes but
insufficient cross-protection.38 The potential absence of cross-
protection coupled with shared antigenicity among reassortants could
result in antibody-dependent enhancement (ADE), a process that likely
contributes to severe secondary DENV infections.41 Therefore, as
OROV and its reassortants coexist, investigating their serological
relationship is crucial to gauge potential ADE risks and any limitations
in cross-protection.41
Bibliographic analysis of publications on OROV
Literature on OROV is scarce. Only 192 peer-reviewed papers
published since July 1, 1961, to June 30, 2023, were retrieved from
PubMed, highlighting the sparse research interest in OROV (figure
3A). We also conducted a Boolean search on the Web of Science on
March 31, 2023, using the terms: “OROV” OR “virus del oropuche”
OR “Oropouche virus” OR “Oropouche” OR “Oropuche” OR “Iquitos
virus” OR “Madre de Dios virus” OR “Perdoes virus” (Title), OR
“OROV” OR “Virus del oropuche” OR “Oropouche virus” OR
“Oropouche” OR “Oropuche” OR “Iquitos virus” OR “Madre de Dios
virus” OR “Perdoes virus” (Abstract), AND NOT poecilla OR guppy
(Abstract) AND NOT trinidad (Abstract).

3
The search revealed that OROV research is conducted by a small
number of groups; the top five authors contributed 67 articles, which
represent 35% of all the articles on the subject. Further breakdown of
these data indicates that most of these articles come from Brazil (57%)
and the USA (38%). Bibliographic analysis of OROV literature
showed high bibliographic coupling (two given works sharing a
common reference) for articles published in the past decade (figure
3B). High bibliographic coupling can be explained by the low numbers
of publications and little heterogeneity in literature, with the few
researchers working on OROV citing the same papers because of
insufficient new evidence.
Geographical distribution of OROV and its reassortants
Since its initial detection in 1955 in Trinidad and Tobago,42 OROV has
been detected across Central and South America, spanning 5000 km
south and 2000 km west, and its incidence has increased over the past
several decades (figure 4). As of June, 2023, reports of OROV in
human and non-human hosts have emerged pre- dominantly from rural
and forest areas in Brazil,26,28,29,44–65 Peru,66–73 Ecuador,74,75,76,77
Argentina,43 Bolivia,74 Panama,26 Colombia,78–80 and Venezuela.37 The
virus’ first Caribbean detection—after its initial discovery in Trinidad
and Tobago in 1955—occurred in Haiti in 2014,81 followed by a 2016
outbreak in non-endemic coastal regions of Ecuador and Peru76,82 and
its emergence in a densely populated urban zone in northeastern
Brazil.63 In 2020, nearly half of a small rainforest village in French
Guiana experienced a dengue-like syndrome, with OROV diagnosed
in 43% (41/95) of inhabitants, suggesting OROV can present with high
attack rates.83 Colombian data from 2019 to 2021 attribute 11%
(87/791) of acute febrile disease during this time period to OROV, with
16% (92/568) of Colombian patients testing positive for OROV IgG
antibodies in chemiluminescent immunoassays.84 Altogether, the
available literature suggests a wide geographical expansion of OROV.
IQTV was first isolated in 1999 from a patient in Iquitos, Peru, and has
caused at least 160 human cases between 2005 and 2006.38 In 2006,
15·4% (160/1037) of patients with undifferentiated febrile illness from
Iquitos showed IQTV neutralising antibodies in plaque reduction
neutralisation tests, which is slightly higher than the 14·9% (154/1037)
found for OROV. Additionally, 3·4% (35/1037) had antibodies to both
viruses, suggesting cocirculation or substantial cross-neutralisation.38
MDDV was identified in a febrile patient in Peru39 in 2007, and in a
white-faced capuchin monkey in Venezuela in 2010, nearly 2000 km
away.37 PDEV has only been isolated once in 2012, from two black-
tufted marmosets in Minas Gerais, Brazil.29
Clinical presentation, treatment, and vaccine
Most information available on the clinical presentation of the infection
with OROV and its reassortants stems from case reports or small-scale
outbreaks. Therefore, the evidence available on clinical presentation
and sequelae is poor, and morbidity and mortality could be
underestimated.
Oropouche fever is generally mild and self-limiting and often begins
with a 3–10-day incubation period,44,62 after which patients develop a
febrile illness with headache, arthralgia, myalgia, nausea, vomiting,
chills, and photophobia.72,82,83,85 Some patients have less common
symptoms, such as rash, retro-orbital pain, anorexia, and haemorrhagic
manifestations.44,72,82 Severe clinical presentations, which are rare,
could involve CNS infections that result in aseptic meningitis or
meningoencephalitis.57,62,64,83,86,87 The disease is typically biphasic,
with an acute phase lasting 2–4 days, followed by a remission and a
resurgence of symptoms 7–10 days after onset.64,83 Most patients
recover without sequelae, although persistent myalgia and asthenia
lasting up to 1 month have been reported.83 No deaths have been
attributed to OROV infection yet. Similarly to OROV, IQTV infection
presents with fever, headache, eye pain, body pain, arthralgia,
diarrhoea, and chills.38 MDDV was reported to cause undifferentiated
febrile illness, and no human infection has been recorded for
PDEV.29,39 The rate of asymptomatic infections is difficult to estimate
as studies generally include febrile patients; a single study reported
symptoms in up to 63% of patients with OROV infection.65
Co-infections of OROV with reassortants have not been reported.
However, acute infections of OROV and other arboviruses can occur,
as observed in Peru and Colombia with DENV.72,73,84 Co-infections of
OROV–ZIKV and OROV–CHIKV have been described at low
detection rates,72 as well as a triple co-infection of OROV–DENV–
CHIKV identified once in western Brazil,86 but the effect of co-
infections by OROV and other co-circulating arboviruses on disease
severity remains unclear.
Currently, no specific antiviral treatment against OROV infection is
available. Ribavirin, a broad-spectrum antiviral, was ineffective
against OROV in vivo in mice;88 mycophenolic acid, which has shown
in vitro activity against yellow fever virus, DENV, and Semliki Forest
virus, was also ineffective against OROV.89,90 Interferon alfa can
restrict viral replication in vitro and in vivo in mice when administrated
early or pre-infection, but its clinical relevance is unclear.91
Although no vaccine is currently available for OROV, a single study88
presented a candidate vaccine based on a replication-competent
chimeric vesicular stomatitis virus expressing complete or truncated
OROV glycoproteins, which showed a reduction in viral loads and
symptom severity in mice; moreover, another study predicted novel
epitope candidates for epitope-based peptide vaccine design against
OROV using computational methods.92 Other orthobunyavirus
vaccines could guide the development of OROV vaccines. A
chemically inactivated Schmallenberg virus (SBV) vaccine is licensed
for veterinary use in the EU,93 and an SBV nucleoprotein fragment-
based vaccine has been shown to reduce viraemia in infected mice.94
Evidence regarding vaccine-induced cross-protection in the Simbu
serogroup is scarce. A trivalent vaccine comprising two inactivated
Simbu serogroup viruses, Akabane virus (AKAV) and Aino virus
(AINOV), failed to prevent SBV infection in cows.95
Laboratory diagnosis
Identifying the causative agent of acute febrile illness without
laboratory tests is hardly feasible and sometimes inaccurate. Therefore,
many treatable diseases with non- specific symptoms are misdiagnosed
as dengue or malaria.7,96 Similarly, discrimination between OROV and
other pathogens causing acute febrile illness based solely on clinical
symptoms was shown to be unreliable.73 Consequently, patients are
being misdiagnosed or untreated and outbreaks could remain
unrecognised. To verify acute OROV infections, laboratories typically
employ three primary diagnostic criteria: a positive PCR result, the
identification of specific IgM, or the detection of seroconversion
through paired samples. Historically, serological methods were
predominant but have been gradually replaced by molecular
methods.24,27,40,85,97
Molecular diagnosis
Molecular detection of OROV is typically possible during the first
week on acute-phase specimens.24,40,61,85,98 Real- time RT-PCR in sera
collected during the first 5 days of illness has shown a 93% detection
rate.98 Serum viral loads can range from 10⁴ to 10⁸ genome copies per
mL,98 and the presence of low viraemia in recent cases highlights the
need for sensitive PCR assays.84 Real-time RT-PCR is preferred for
early-stage diagnosis due to its speed, ease, and reduced contamination
risk. Various in-house tests have been developed,51,61,77,84,98,99,100
including multiplexed formats for detecting co-circulating Mayaro
virus.101 Most assays target the OROV S segment,77,98,100,101 which does
not distinguish between OROV and its reassortants. Typing can be
enabled by including M segment-based primers.61,84,102 Conventional
RT-PCR is widely used for research51,61,77,98,99,100,102 alongside virus
isolation.76,79,83
Serum or plasma are the preferred samples for OROV
diagnosis.51,54,72,79,83,84 In patients with OROV infections involving the

4
CNS, viral RNA detection, viral isolation from cerebrospinal fluid
(CSF), and IgM detection in CSF have been reported.57,62,64
Furthermore, OROV has been detected by PCR in saliva and urine
collected from symptomatic patients within 5 days of disease onset,61,63
suggesting that these samples could serve as alternative specimens for
OROV detection during the acute phase of infection. Additionally,
although impractical for individual patient testing due to high cost and
reduced sensitivity, metagenomic sequencing could serve as an
additional environmental surveillance tool. Pooled vector-derived or
patient-derived samples can provide genetic information of circulating
virus strains. The use of samples from wastewater has also been
explored for flaviviruses,103 but further exploratory analysis is needed
to assess whether this approach can be employed for other arboviruses,
especially OROV. Further implementation of these methods will help
to clarify OROV genetic diversity, enabling the improvement of
diagnostic assays, vaccines, and antivirals.103,104
Serological diagnosis
After acute infection, molecular testing should be supplemented by
serological methods. Immune responses against OROV are not well
studied, but serological procedures can detect OROV-specific IgM and
IgG in serum, plasma, and CSF.24,40,56,59,64,67,71,84,85 Ideally, IgM testing
in CSF should be done together with IgM testing in paired serum
samples and supported by assessments of blood–brain barrier leakage
to confirm intrathecal immunoglobulin synthesis.105
Existing in-house serological tools include enzyme immunoassays,
neutralisation tests, complement fixation tests, immunofluorescence
tests, and haemagglutination inhibition tests.74,75,28,42,45,48,70,106 Tests
based on the nucleocapsid (N) protein, which elicits a strong humoral
immune response, have been developed;84,107,108 however, these tests
show cross-reactivity due to the conservation of epitopes on N proteins
among many Simbu serogroup viruses.107–110 Although commercial Gc
or N antigens are not available for OROV, robust commercial immuno-
assays are hardly available. Neutralisation tests are considered the gold
standard for arbovirus serology,13 but they are not often used for patient
diagnosis due to long turnaround times and the need for handling
infectious viruses under biosafety level 3 conditions.
Robustness of laboratory diagnosis in articles reporting infection
with OROV and its reassortants
55 articles published between 1960 and 2022 indicate a shift in
diagnostic tools used for OROV and reassortant infections (figure 5).
Before the 2010s, serological methods were commonly used,
particularly in retrospective studies.28,45–50,53,68,70,106 However, in the
past 15 years, the use of robust techniques (eg, RT-PCR and
sequencing) has greatly enhanced the accuracy of detecting OROV
infections and differentiating them from their reassortants.29,38,39,44,57,60–
64,66,67,72,76,79,81–83,86 Nonetheless, OROV and its reassortants are most
probably still underdiagnosed or misdiagnosed due to symptoms
resembling those of classical arboviral diseases reported in Central and
South America, highlighting the need for effective differential
laboratory testing.72
Transmission cycles of OROV
Understanding the arboviral transmission cycle allows for the
identification of geographical areas and people at risk for infection.
OROV is probably maintained through a sylvatic and an urban cycle
(appendix p 1). The biting midge Culicoides paraensis is considered
the main OROV vector because of viral isolation during outbreaks and
vector competence observed in vivo under laboratory conditions.46,47–
49,65,111 Humans are the suspected link between the sylvatic and urban
transmission cycles given that C paraensis is present in both urban and
rural settings.40 Some OROV outbreaks have also coincided with
human-driven environmental changes, such as deforestation, increased
agriculture, and infrastructure development.69,82,83,112 These changes
can alter vector distribution and increase contacts among vectors,
vertebrate reservoirs, and humans.113
Humans are the major vertebrate hosts in the urban cycle of
OROV.38,68,71 Whether urban OROV transmission is transient,
introduced via human movements, or permanent but at low levels and
therefore undetected is unclear, and the potential role of domestic
animals as amplifying hosts requires further investigation.34,46,102,114
The sylvatic cycle is less understood than the urban cycle, with
definitive reservoirs and amplifying hosts yet to be identified. OROV
antibodies have been found in wild birds, sloths, non-human primates,
and rodents,46,47,78,115,116,117 alongside sporadic isolations from sloths
and non-human primates in Brazil.45,53,102 Systematic studies of OROV
non-human reservoir hosts are scarce.
The detection of OROV in a coastal region of Ecuador where
C paraensis is absent (or yet to be found118) suggests the possibility of
other vectors involved in OROV transmission.76 Minor mutations can
enhance vector- borne transmission, as evidenced by CHIKV adaption
to Aedes albopictus.119 Culex quinquefasciatus, a primary and
widespread vector of West Nile virus,5 was suspected to be involved in
OROV transmissions in Brazil and French Guiana.44,83,120
Conversely, low efficiency in viral transmission has been observed
experimentally for C quinquefasciatus.121–123 The main DENV vectors,
Aedes aegypti and A albopictus, have been found to be ineffective
OROV vectors,121 whereas other mosquitoes belonging to the species
Coquillettidia venezuelensis,42 Aedes serratus,42,45,124 Psorophora
cingulata, and Haemagogus tropicalis were found to be naturally
infected by OROV.124 However, vector competence studies for these
mosquito species are missing. If confirmed, this wide vector range
would be only comparable with few other clinically relevant
arboviruses, such as Japanese Encephalitis virus and Rift Valley fever
virus.5 Although OROV dissemination seems restricted to South and
Central American regions, both primary and supposed secondary
vectors are not,122,125 highlighting the risk of further dissemination and
emergence in other parts of the world. A study estimated that 2–5
million people were at direct risk of exposure to OROV in regions up
to the southern USA.126
Animal models
To date, hamster and mouse models have been described for OROV
infection but not for reassortants. In immunocompetent Syrian golden
hamster models, OROV causes systemic disease analogous to humans,
as well as liver injury and severe neurological symptoms, such as motor
deficits and paralysis.127,128 In immuno- competent mice, OROV
infection outcomes range from no clinical signs, to few,88,129–131 and to
low mortality rates.132 OROV infection in mice lacking the receptor for
type I interferon (IFN) induces increased mortality rates and caused
extensive liver and spleen damage.129,130 Suckling mice develop severe
neurological symptoms before death with no observed
hepatotropism.89,91,133,134 OROV hepatotropism has been observed in
naturally infected non-human primates, with OROV and PDEV
isolated from marmosets,29,135 but non-human primate models that
better characterise infection by OROV and reassortants have not yet
been developed.
Pathogenesis
OROV infection elicits varied cellular and systemic responses,
involving multiple host factors. Peripheral blood mononuclear cells are
supposed initial targets of OROV, but their virus replication capacity
is relatively small.136 Plasmacytoid dendritic cells important for type I
IFN and proinflammatory cytokines production, are also probable
targets.130
A key aspect of OROV immune evasion is its NSs protein, which
antagonises IFN and could induce apoptosis as observed in other
pathogenic orthobunyaviruses, sidestepping immune responses.137,138
Additionally, microRNAs miR-217 and miR-576–3p increase during
infection, which potentially inhibits the antiviral IFN beta response.139

5
For cellular entry, lipoprotein-related protein 1 is a key host factor
aiding OROV entry, as it is also for Rift Valley fever virus,
highlighting its role in bunyavirus infections.140
In severe cases, OROV can affect the CNS; however, the exact
infiltration mechanism remains unclear. An in vivo mouse study
indicated blood–brain barrier leakage and a possible neural pathway
for initial CNS invasion.134 OROV disrupts IFN responses in human
primary astrocytes in vitro, potentially leading to neuropathology.141
However, ex vivo studies in adult human brains found no OROV-
infected astrocytes, whereas microglia were more commonly
infected.142
Gaps of knowledge and recommendations
Genetics and reassortants
No instances of OROV reinfection have been recorded, and examining
the serological relationship between OROV and its reassortants
remains crucial. Similarly, the impact of reassortment on OROV host
range and pathogenesis remains unknown. We recommend that in vitro
investigations are conducted to explore the potential for reassortment
between OROV and other American orthobunyaviruses. Furthermore,
orthobunyavirus surveillance capabilities should be enhanced to
distinguish and probe for reassortant viruses.
Disease surveillance and epidemiology
OROV surveillance is suboptimal, with incomplete diagnostic panel
inclusion of the virus and its reassortants. Clinical understanding is
based on minor outbreaks or isolated cases, necessitating longitudinal
studies to describe asymptomatic infection and define clinical
presentation and potential long-lasting sequelae. The correlation of
specific symptoms or their severity with OROV and reassortant
infection also requires further investigation. Existing seroprevalence
studies are scarce. Consequently, reliable data to estimate the true
extent of OROV circulation or to distinguish it from its reassortants are
currently lacking. We recommend that comprehensive longitudinal
studies and cross-sectional seroprevalence studies are implemented to
improve understanding of eventual long-lasting or chronic effects of
OROV fever, and to monitor the circulation of OROV and its
reassortants to assess the potential for reinfection or multiple infections
by different OROV reassortants. Metagenomics combined with vector
or patient pool- based analyses can improve genomic surveillance and
allow investigation of co-infections and vectors. The potential of
wastewater surveillance for the detection and monitoring of OROV and
other circulating arboviruses should also be explored.
Entomological and environmental surveillance
Despite isolating OROV from various species, the role of mosquitoes
in transmission cycles remains unclear.121 Should OROV better adapt
to the prevalent vectors A aegypti and A albopictus, its geographical
spread could be substanially widened and, in regions where OROV is
already present, infections could increase.119 The role of C
quinquefasciatus, given its wide geographical spread, needs
clarification.120 More vector competence studies are therefore
necessary. Furthermore, information on OROV’s transmission cycle is
either outdated or incomplete. Virus isolation from reservoirs has been
sporadic; only a handful of articles report isolation of OROV53,135 or
reassortants37,135 from non-human hosts. Additionally, the
dissemination of these hosts does not completely coincide with regions
reporting OROV outbreaks. For example, the sloth and capuchin
species, from which OROV and MDDV have been isolated, are native
solely to northwestern South America143,144—far below the
geographical range of described OROV infections. Surveillance of
sylvatic reservoirs and emergence events needs to be increased, and the
potential for long-distance OROV dissemination (eg, by migratory
birds) requires investigation. We recommend that currently known
sylvatic reservoirs are confirmed and that further reservoirs and
amplifying hosts are identified, especially in regions with outbreaks
within the past decade or in regions with an apparent absence of known
reservoirs.
Detection tools
The evaluation of OROV diagnostics remains difficult due to the
absence of reference material, external quality assessments, and
comparative studies. Information about viral loads and immune
response kinetics in various body fluids stems only from isolated cases.
Longitudinal studies are essential to identify optimal specimens for
diagnosis and standardise sampling for diagnostics and research.
Additionally, exploring potential correlations between prolonged
viraemia or high viral loads and disease severity, akin to other
arboviruses, could be valuable.145 Current molecular and serological
assays often struggle to differentiate between OROV and its
reassortants, and commercial tests are mostly unavailable. Laboratory
diagnostics of OROV should therefore be improved. The development
of point-of-care rapid diagnostic tools could help implement OROV
diagnostics on a larger scale, especially in rural areas. Pathogenesis,
animal models, and cross-protection OROV pathogenesis is poorly
understood, and factors leading to severe disease remain to be
identified. Reverse genetics systems developed for OROV could be
exploited for this purpose.137,146 Animal models are restricted to
rodents, but the development of adequate animal models will be
essential for OROV pathogenesis and vaccine research, including
assessments of potential ADE. Safe human challenge models have
proven to be valuable for DENV research and could be explored for
OROV in the future.147 The amino acid divergence in the M segment
between OROV and IQTV or MDDV resembles that of other Simbu
serogroup viruses, for which an experimental vaccine failed to afford
cross-protection. The available genomic and serological data for
OROV and its reassortants suggest that development of vaccines
against Oropouche fever could be challenging. We recommend that
animal models for OROV and reassortants are expanded, that the cross-
protection potential of existing vaccines be explored for OROV, and
that the development of an effective, safe, stable, and affordable
OROV vaccine is prioritised.
Conclusion
OROV has the potential to emerge as a substantial threat given its wide
host and vector range, diverse environmental spread, potential for
severe course of disease, and existence of human-infecting reassortants
that could correspond to different serotypes. Despite the promising
increase in research, knowledge of the virus lags compared with other
American arboviruses. Intensifying international collaboration,
including between Latin American countries in which OROV is
endemic, and funding to bridge these knowledge gaps is imperative, as
this will simultaneously enhance our understanding of other neglected
diseases and inform public health policies. Therefore, OROV
represents a prototypic NTD that necessitates investigation to assess
and minimise its health burden.
Contributors
KMW, IP-H, and LP contributed to the literature search, figure conceptualisation and
realisation, and writing of the original draft. EFdO- F and CF contributed to figure
conceptualisation and data analysis. XdL contributed to t he study design. JFD contributed to
the study design and editing and writing of the final dra ft. All authors were involved in
reviewing the manuscript.
Declaration of interests
We declare no competing interests.
Acknowledgments
This study was supported by the Deutsche Forschungsgemeinschaft project COALITION
(project number DR 810/6–1), t he European Union’s Horizon 2020 Research and Innovation
programme through the ZIKAlliance pro ject (grant number 734548), and the Ho st Switching
Pathogens, Infectious Outbreaks and Zoonosis (HONOURS) innovative training network
(grant number 721367).

6
Figure 1: OROV structure and genomic sequence availability
(A) OROV structure and genomic organisation. (B) Nucleotide sequences available on GenBank for each segment up to June,
2023. A total of 15 sequences (segments: small [S], n=11; medium [M], n=2; and large [L], n=2) labelled “unverified” on
GenBank (ie, GenBank staff could not verify the accuracy of the submitted sequences) were excluded. For S and L segments,
sequences from OROV and reassortants are merged; for the M segment, sequences from OROV and reassortants are
represented separately. Information on the genomic sequences used for figure 1B is available in the appendix. IQTV=Iquitos
virus. MDDV=Madre de Dios virus. NSm=non-structural protein M. NSs=non-structural protein S. OROV=Oropouche virus.
PDEV=Perdões virus. RdRp=RNA-dependent RNA polymerase. UTR=untranslated region. Figure created with BioRender.com
(A) and R ggplot2 package (B).

7
Figure 2: Evolutionary characteristics of OROV and genetically related viruses
(A) Maximum amino acid sequence distance between OROV, selected members of the Simbu serogroup, and BUNV belonging
to a different serogroup. Sequences were aligned using MAFFT (https://mafft.cbrc.jp/; algorithm: auto; scoring matrix
BLOSUM62) with all available complete genomic sequences of OROV, IQTV, MDDV, and PDEV, and to the reference sequences
of FPV, JATV, MANV, SBV, SIMV, and UTIV. These viruses were selected to improve visualisation of the genetic diversity
between OROV and its reassortants by comparing the distance within Simbu and between Simbu and Bunyamwera
serogroups. The amino acid p-distance matrix was generated using MEGA X (www.megasoftware.net) using a pairwise
deletion option. JATV, originally considered an OROV reassortant,35 is now classified as a separate species by the International
Committee on Taxonomy of Viruses.36 Sequences labelled “unverified” in GenBank (ie, GenBank staff could not verify the
accuracy of the submitted sequences) were excluded. All sequences were retrieved from GenBank in June, 2023. (B)
Maximum likelihood phylogenetic trees done in MEGA (MEGA X) using a Whelan and Goldman substitution model and
complete deletion option based on translated coding sequences of the L, M, and S segments. Black circles at nodes represent
support values of at least 0·70 from 1000 bootstrap replicates. OROV sequences were selected to cover the variability of the
three genome segments and all available sequences of the reassortant viruses. Information on the genomic sequences used
for figure 2A and 2B is available in the appendix. OROV=Oropouche virus. L=large segment. M=medium segment. S=small
segment. IQTV=Iquitos virus. MDDV=Madre de Dios virus. PDEV=Perdões virus. JATV=Jatobal virus. UTIV=Utinga virus.
FPV=Facey’s Paddock virus. MANV=Manzanilla virus. SBV=Schmallenberg virus. SIMV=Simbu virus. BUNV=Bunyamwera virus.
AUS=Australia. BRA=Brazil. ECU=Ecuador. NLD=Netherlands. PER=Peru. TTO=Trinidad and Tobago. VEN=Venezuela.
ZAF=South Africa.

8
Figure 3: Literature on Oropouche virus (OROV) and its reassortants
(A) The number of publications in PubMed on OROV and reassortants up to June 30, 2023. (B) Bibliographic coupling in OROV
literature from a Web of Science search (accessed March 31, 2023). Each node represents a publication (indicated with name
of the first author and year of publication); connected nodes represent articles sharing at least 15 references. Nodes are
distributed in two dimensions by the number of references shared; clustered nodes therefore represent similar publications.
Node size represents the total link strength of the node (ie, the sum of all link scores from each publication pair). The bigger
the node, the higher the bibliographic coupling with the rest of the reference set. Colour scale represents the year of
publication. Figure created with RStudio (A) and VOSviewer (B; University of Leiden, 2010).

9
Figure 4: Oropouche virus detections in humans, other vertebrate hosts, midges, and mosquitoes
Confirmed cases were detected with sequencing or RT-PCR or neutralisation tests or viral isolation and complement fixation
test or seroconversion in paired samples with any type of serological tool. Probable infections were detected with ELISA,
immunofluorescence tests, or haemagglutination inhibition tests in non-paired samples. Estimated cases were taken from
the literature without laboratory evidence. Cases from Argentina in 2000–09, included in the map, were reported as personal
communication in a textbook of paediatric infectious diseases43 and not in a peer-reviewed article. Host and vector cases
detected with any method were included. Maps were created with QGIS (https://qgis.org/en/site/; long-term release version
3.22 Białowieża) on a regional level and by decade. Supporting information regarding the infections is available in the
appendix. IQTV=Iquitos virus. MDDV=Madre de Dios virus. PDEV=Perdões virus.

10

11
Figure 5: Robustness of laboratory diagnosis of human infections with Oropouche virus and reassortants
(A) Predominance of serodiagnosis and viral isolation. (B) Widespread use of molecular detection and sequencing. For both
panels, confirmed cases were detected with sequencing, RT-PCR, neutralisation tests, viral isolation combined with CF, or
seroconversion in paired samples with any type of serological tool. Probable cases were detected with ELISA,
immunofluorescence assays, or haemagglutination inhibition tests on a single sample. Studies with no black dots are based
solely on symptoms. CF=complement fixation. *Studies include different study groups, presented separately. ¤ Describes
cases of Iquitos virus. # Describes cases of Madre de Dios virus. For clarity of presentation only one surname is shown.
Details on the studies summarised are available in the appendix.
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