The genus Alphavirus in the family Togaviridae contains several mosquito-borne viruses that are maintained in sylvatic amplification cycles involving wildlife and forest-dwelling mosquitoes, such as Chikungunya virus (CHIKV) and Venezuelan equine encephalitis virus (Chen et al., 2018). While some viruses cause sporadic spill-over infections and local outbreaks in humans and domestic animals, others adapt efficiently to anthropophilic mosquito species and humans as amplification hosts, enabling spread to new geographic regions causing millions of infections worldwide, as is the case for CHIKV (Weaver et al., 1996; Weaver, 2014). According to the International Committee on Virus Taxonomy (ICTV), the genus Alphavirus contains more than 30 virus species, grouped into two monophyletic clades: (i) Old World alphaviruses, such as CHIKV, which can induce rash and chronic arthritis in humans, and (ii) New World alphaviruses, such as Eastern equine encephalitis virus, which can cause neurological disease in humans and horses with high mortality rates (Zacks and Paessler, 2010; Chen et al., 2018). Additionally, two aquatic alphaviruses and several insect-specific alphaviruses have been identified (Weston et al., 1999; Nasar et al., 2012).

Alphaviruses are characterized by small virions, typically 65–70 nm in diameter, and composed of a single capsid protein and three glycoproteins forming spherical, enveloped virions (Cheng et al., 1995; Chen et al., 2018). The genome of alphaviruses consists of 10–12 kb positive-sense, single-stranded ribonucleic acid (ssRNA), including two open reading frames (ORFs) (Chen et al., 2018). ORF1 codes for four non-structural proteins (nsP1-4) and ORF2 for six structural proteins (CP, E1-3, 6 K/TF) (Strauss and Strauss, 1994).

Sindbis virus (SINV), an Old World alphavirus, circulates among ornithophilic mosquitoes and birds in an enzootic transmission cycle (Lundström et al., 2019). SINV shares its main vector, mosquitoes of the genus Culex, with other emerging arboviruses such as West Nile virus (Mcintosh et al., 1976; Francy et al., 1989). Spill-over infections to humans and horses can occur via so-called bridge vectors, although they cannot sustain viral transmission (Lundström et al., 2019). Infection with SINV can cause an acute febrile illness with rash, arthritis, and musculoskeletal symptoms in humans (Skogh and Espmark, 1982; Espmark and Niklasson, 1984; Kurkela et al., 2005; Sem Ouilibona et al., 2018; Didier et al., 2024). SINV is geographically widespread and has been found in Africa, Europe, Asia, and Oceania, also designated as Babanki, Ockelbo, Karelian fever, Kyzylagach, or Sindbis-like virus, depending on the location of detection (Taylor and Hurlbut, 1953; Lundström and Pfeffer, 2010; Adouchief et al., 2016). It was first detected in Egypt in 1952. A total of six SINV genotypes were identified based on E2 gene phylogenetic analyses (Lundström and Pfeffer, 2010; Pickering et al., 2019). In Northern Europe, where the virus was most likely introduced by migratory birds from Central Africa in the 1920s, SINV is endemic and causes disease in humans (Turunen et al., 1998; Ling et al., 2019). While most studies on SINV have been conducted in Finland, Sweden, and South Africa, knowledge regarding its geographic distribution and incidence in other regions in Africa remains limited.

Middelburg virus (MIDV), another Old World alphavirus, was first isolated from Aedes caballus and Aedes (Banksinella) sp. mosquitoes in South Africa in 1957 and has subsequently been detected in Zimbabwe, Cameroon, Kenya, Senegal, and the Central African Republic (Kokernot et al., 1957; Attoui et al., 2007; Tricou et al., 2014). In southern Africa, MIDV has been found in livestock and wildlife, causing fever and encephalitis, but has not been associated with larger outbreaks (van Niekerk et al., 2015; Steyn et al., 2020).

Uganda is a hotspot region for arbovirus circulation and emergence, including several arboviruses that have been detected for the first time in Uganda’s forests, such as West Nile virus and Zika virus (Smithburn et al., 1940; Dick et al., 1952). This study aimed to sample mosquitoes from pristine and remote areas in Uganda that have an extraordinary amount of biodiversity (Convention on Biological Diversity, 2016). The objective was to assess the genetic diversity of alphaviruses in mosquitoes within enzootic amplification cycles in Uganda that have so far been neglected. Such explorations are crucial for detection and control of circulating (endemic) arboviruses, as well as to monitor genetic and phenotypic changes contributing to arbovirus emergence.

Arbovirus surveillance in hotspot regions of arbovirus emergence provides information on circulating strains and is important for correct diagnosis and disease control. Here, we collected over 23,000 mosquitoes in a lowland tropical rainforest, in a savannah gallery forest, and adjacent rural communities in southwestern Uganda and tested them for infection with alphaviruses. We detected SINV and MIDV in individual mosquitoes from two different primary habitat types, suggesting endemic circulation of these viruses in Uganda.

To our knowledge, this is the first detection of MIDV in Uganda. MIDV was found in Semuliki National Park and in Queen Elizabeth National Park. The presence of MIDV in two National Parks with distinct ecosystem types (lowland tropical rainforest and a savannah-like gallery forest) suggests endemic maintenance, and that MIDV has been circulating unnoticed for an unknown period in Uganda. So far, MIDV was associated with Aedes spp. mosquitoes as vectors (Jupp and Cornel, 1987). Here, we identified Mansonia africana and Eretmapodites intermedius mosquitoes as potential further MIDV vector species. Most mosquitoes of the genus Eretmapodites are sylvatic, while a few species have adapted to the disturbed environment in rural communities and breed in artificial containers such as the Eretmapodites chrysogaster group, which includes Eretmapodites intermedius (Someren, 1949; Lounibos, 1978). Birds and mammals serve as blood meal sources (Musa et al., 2020). Mansonia spp. mosquitoes are active at dawn and dusk, searching for a variety of hosts including birds, mammals, and reptiles (Galardo et al., 2022). Our understanding regarding host range and pathogenicity is limited. Experimentally infected sheep showed viremia and pyrexia, and infection of newborn mice was shown to be lethal (Kokernot et al., 1957). Horses can develop severe symptoms, including neurologic disease, sometimes with fatal outcome, as observed in Zimbabwe in 1993 and in South Africa in 2015 (Attoui et al., 2007; Van Niekerk et al., 2015). Antibodies against MIDV have been detected in humans and a recent study detected MIDV genome copies in cerebrospinal fluid and whole blood samples of patients with neurological symptoms suggesting that MIDV may cause disease in humans (Smithburn et al., 1959; Fourie et al., 2022). The efficient and rapid growth of the Ugandan MIDV isolate on the tested human and livestock cell lines suggests no overt impairment of the potentially enzootic isolate with respect to host range. However, future investigations aiming to identify MIDV in acute febrile patients and animals, as well as testing for the presence of neutralizing antibodies against MIDV in humans and animals, would be needed to shed light on the geographic distribution and burden of MIDV in Uganda.

The detection of SINV in a Culex Culex sp. mosquito from Semuliki National Park confirms previous findings of SINV in Uganda, where it was first detected in Coquillettidia fuscopennata mosquitoes in Buwaya near Entebbe in 1960 and shortly afterwards in humans in 1961 (Haddow, 1961/1962; Woodall et al., 1964). SINV has been found in Culex sp. and Mansonia sp. mosquitoes from Semuliki National Park and Lake Mburo National Park in 2010 (Mossel et al., 2017). Furthermore, SINV neutralizing antibodies were detected in bats from Kampala, suggesting that the virus occurs across the country (Kading et al., 2018). Across sub-Saharan Africa, SINV has been found in various mosquito species, including Culex quinquefasciatus from Burkina Faso, Culex cinereus from the Central African Republic, and Culex univittatus, known as main vector for SINV in countries such as South Africa and Kenya (McIntosh and Jupp, 1979; Sigei et al., 2018). We could not unambiguously identify the vector species as the mosquito species could not be identified on a morphological basis and its COI gene sequence showed only a maximum relationship of 97.5% to Culex watti. Culex watti has not yet been described as a vector for SINV. Although SINV was isolated from an enzootic vector, it is likely that it can readily infect humans and equids, as supported by cell culture infection experiments and phylogenetic analyses. SINV MP762-UG-2019 demonstrated rapid replication rates across all tested cell lines and was shown to belong to genotype-I, that has been reported from Africa, Europe, and the Middle East and is associated with febrile illness (Skogh and Espmark, 1982; Espmark and Niklasson, 1984; Kurkela et al., 2005; Sem Ouilibona et al., 2018; Didier et al., 2024). The symptoms are usually self-limiting and clear spontaneously within a few weeks, but in approximately 25% of patients, the symptoms can persist for years (Niklasson et al., 1988; Kurkela et al., 2005). Outbreaks were mainly reported from northern Europe, mainly Sweden and Finland, and sporadically from South Africa (Espmark and Niklasson, 1984; Jupp and Meenehan, 1986; Suvanto et al., 2022). For example, the incidence of SINV in Finland peaked with 566 reported cases in 2021, resulting in the most extensive outbreak of a mosquito-borne disease in Europe that year (Suvanto et al., 2022). As is the case for MIDV, surveillance for and diagnosis of SINV infection is limited in Uganda, and knowledge of human infection rates is unknown.

Like Chikungunya virus (CHIKV), MIDV and SINV are categorized as Old World alphaviruses, traditionally associated with milder disease compared to New World alphaviruses like the equine encephalitis viruses. Eastern equine encephalitis virus, along with Venezuelan and Western equine encephalitis virus, can lead to severe neurological disease with high mortality rates in both horses and humans (Meyer et al., 1931; Deresiewicz et al., 1997). The characterization of Old World alphaviruses as less severe warrants re-evaluation, given that they also induce significant clinical manifestations in humans and animals. For instance, CHIKV is associated not only with chronic arthritis but also with neurological manifestations in humans (Rampal and Meena, 2007). SINV and MIDV have also been linked to neurological symptoms in horses and other mammals (Van Niekerk et al., 2015; Steyn et al., 2020).

We observed inconsistencies in the placement of MIDV MP61-UG-2019 and MP1299-UG-2019 in phylogenetic analyses. In phylogenetic analyses based on concatenated ORF sequences and the non-structural polyprotein, both MIDV strains were placed as sister taxa in a clade with sequences from South Africa (Figure 3A). Phylogenetic analysis based on the structural polyprotein placed MP61-UG-2019 and MP1299-UG-2019 in basal relationship to all other sequences (Figure 3B). This inconsistency may originate from a recombination event between Semliki Forest virus and Mayaro virus within the E1 gene, as observed previously (Attoui et al., 2007). The basal position and the detection of the two strains in mosquitoes from primary ecosystems support an origin from sylvatic (enzootic) amplification cycles. The exploration of genetic and phenotypic traits of sylvatic variants helps to identify changes in emerging epidemic variants, as documented for CHIKV and VEEV (Wang et al., 1999; Powers et al., 2000). Especially CHIKV demonstrated a high epidemic potential and how fast a new emerging epizootic variant can spread geographically. In the last 15 to 20 years CHIKV has spread to new territories all over the world and is responsible for millions of human infections (Weaver, 2014; Wahid et al., 2017). Similarly, SINV showed that a solitary dispersal event can result in the establishment of a virus in a new geographic region (Ling et al., 2019), given conditions such as the availability and abundance of vector, host, and reservoir under appropriate climate conditions. It has been suggested that SINV-I was introduced to Sweden from central Africa via migratory birds in the 1920s and subsequently spread to central and southern Europe in the 1960s and 1970s (Ling et al., 2019). Particularly biodiversity loss, changes in land use, and climate have been suggested to facilitate the spread of viruses to new geographic regions (Institute of Medicine Forum on Microbial T, 2008).

This study highlights the importance of arbovirus surveillance in hotspot regions of arbovirus emergence for understanding the genetic diversity of circulating arboviruses and for the implementation of respective diagnostics and disease control. We identified the endemic circulation of SINV and MIDV in Uganda, as well as provided the first detection of MIDV in the country. Both viruses infect humans and MIDV also causes disease in livestock. The potential impact of these viruses on human and animal health is currently unknown as no seroprevalence studies have been conducted in the region. Clinical data from patients with compatible symptoms are also not available. It is likely that MIDV and SINV infections are underdiagnosed. However, the geographic distribution of SINV and MIDV in Uganda would need to be investigated for more thorough risk assessments with respect to human and animal health. It may also be the case that both viruses are mainly restricted to the sylvatic environments in Uganda. The isolation of SINV and / or MIDV from epizootic amplification cycles in Uganda could add knowledge regarding virus adaption and evolution during emergence processes from sylvatic amplification cycles. This study further raises questions regarding the pathogenicity, host range and amplification cycles of these viruses, particularly of MIDV, which has been less studied.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.

SG: Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. GE: Investigation, Writing – review & editing. JO: Investigation, Writing – review & editing. TJ: Methodology, Writing – review & editing. AN: Methodology, Resources, Writing – review & editing. JL: Methodology, Resources, Writing – review & editing. IR: Methodology, Resources, Writing – review & editing. SJ: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing.