Detection of two alphaviruses: Middelburg virus and Sindbis virus from enzootic amplification cycles in southwestern Uganda

Abstract

Our knowledge of alphavirus genetic diversity is mainly based on viruses isolated from anthropophilic mosquito species, humans, and livestock during outbreaks. Studies on alphaviruses from sylvatic amplification cycles in sub-Saharan Africa have been conducted less often than from epizootic environments. To gain insight into alphavirus diversity in enzootic transmission cycles, we collected over 23,000 mosquitoes in lowland rainforest and savannah gallery forest in southwestern Uganda and tested them for alphavirus infections. We detected Sindbis virus (SINV) in a Culex Culex sp. mosquito and Middelburg virus (MIDV) in Eretmapodites intermedius and Mansonia africana. MIDV is a mosquito-borne alphavirus that causes febrile illness in sheep, goats, and horses and was previously not known to occur in Uganda. SINV, also a mosquito-borne alphavirus, causes mild infections in humans. Full genomes of SINV and MIDV were sequenced, showing a nucleotide identity of 99% to related strains. Both isolates replicated to high titres in a wide variety of vertebrate cells. Our data suggest endemic circulation of SINV and MIDV in Uganda.

Keywords: Middelburg virus; Sindbis virus; Togaviridae; Uganda; alphavirus; arbovirus; mosquito.

Figure 1

In vitro host range. (A) Growth kinetics of MIDV in mosquito (C6/36), human (HEK-293 T), monkey (Vero E6), rodent (BHK-21), goat (ZN-R) and sheep (Llu-L) cells infected with a multiplicity of infection of (m. o. i) of 0.01. (B) Growth kinetics of SINV in mosquito (C6/36), human (HEK-293 T), monkey (Vero E6), chicken (DF-I) and blackbird (TME-R) cells infected with a m. o. i. of 0.1.

Figure 2

Phylogenetic relationship of SINV strain MP762-UG-2019. The Maximum likelihood (ML) phylogenetic tree of SINV-I is based on the concatenated nucleotide ORF sequences. The virus sequenced in this study is shown in red. SINV strains are labeled by GenBank Accession number, strain, source of isolation, country, and year of collection. The clades are named after Ling et al. (2019). Bootstrap support values are represented by gray (70–90%) or black (>90%) circles at the respective nodes.

Figure 3

Phylogenetic relationship of MIDV strains MP61-UG-2019 and MP1299-UG-2019. The Maximum likelihood (ML) phylogenetic tree of MIDV based on the concatenated nucleotide ORF sequences (A) and the structural polyprotein (B). The viruses sequenced in this study are shown in red. MIDV strains are labeled by GenBank Accession number, strain, source of isolation, country, and year of collection. Bootstrap support values are represented by gray (70–90%) or black (> 90%) circles at the respective nodes.