Table 1.
Patient information for Middelburg virus positive human cases.
Fig 1.
Rooted phylogenetic tree of 347base pair nsP4 fragment produced from Bayesian analysis using BEAST.
Analysisof 33 taxa, model GTR+G+I of alphaviruses is shown. Posterior probabilities >0.7 are shown on major branches. Middelburg virus human positives described in current study indicated with ZRU numbers and blue circles. Alphavirus complexes indicated by brackets; tree is drawn to scale representing the number of nucleotide substitutions per site.
Fig 2.
Rooted phylogenetic tree of 550 base pair E1 fragment produced from Bayesian analysis using BEAST.
Analysis of 29 taxa, model GTR+G+Iof alphaviruses is shown. Posterior probabilities >0.7 are shown on major branches. Middelburg virus human positives described in the current study are indicated with ZRU numbers and blue circles. Alphavirus complexes are indicated by brackets. The tree is drawn to scale representing the number of nucleotide substitutions per site. Full genomes were obtained for ZRU099/17 (EDTA whole blood sample, ad hocveterinary student) and ZRUH399/17 (CSF sample obtained through NHLS from 2 year old child) following virus culturing on Vero cells for both samples and an unbiased next-generation sequencing approach using SISPA-RACE amplification. Assembled contigs for ZRU099/17 and ZRUH399/17 were mapped to MIDV SAE25/11 resulting in full genomes of 11 673 nucleotides (nt) in length, excluding the poly (A) tail. P-distance analysis for full genomes showed the greatest percent identity with the strain isolated from the spleen of the horse from Zimbabwe (MIDV 857), that presented with AHSV-like signs and the South African strain (MIDV SAE25/11) with even greater percentage identities between the two human strains (S4 Table). The concatenated ORF1 and ORF2 p-distances resulted in 99.48% and 99.51% amino acid (aa) identities for ZRU099/17 and ZRUH399/17 compared to MIDV SAE25/11, respectively. Phylogenetic analysis with strong posterior probability support was obtained as follows: the concatenated (ORF1 and ORF 2) and nsP region placed MIDV in its own complex as seen for analysis using nsP4, while analysis using the sP region placed MIDV within the SF complex as seen with analyses involving the E1 fragment (which forms part of the structural proteins) (Figs 3, 4 and 5). Sequences for both ZRU099/17 and ZRUH399/17 contained the 3’UTR repeats that have been identified in MIDV strains isolated from horses but absent from arthropods (S1 Fig) [19–21]. The function of these repeats is still unknown but it has been hypothesized to play a role in virus adaptation to vectors [45]. S5 and S6 Tables show a summary of amino acid and nucleotide changes between currently described and previously published MIDV strains in the ORF’s and UTR’s, respectively. Only positions where changes between MIDV SAE25/11 and other MIDV strains occurred are indicated since MIDV SAE25/11 is a previously identified South African strain. Changes between ZRU099/17 and ZRUH399/17 compared to MIDV SAE25/11 are highlighted in grey.
Fig 3.
Rooted phylogenetic tree produced from Bayesian analysis using BEASTon 7236 base pair non-structural proteins.
Analysis of 31 taxa, model GTR+G+I of alphaviruses is shown. Posterior probabilities >0.7 are shown on major branches. Sequences of human Middelburg virus positives described in the current study are indicated with ZRU numbers and blue circles. Alphavirus complexes are indicated by brackets. The tree is drawn to scale representing the number of nucleotide substitutions per site. (31 taxa, model GTR+G+I) of alphaviruses is shown. Posterior probabilities >0.7 are shown on major branches. Sequences of human Middelburg virus positives described in the current study are indicated with ZRU numbers and blue circles. Alphavirus complexes are indicated by brackets. The tree is drawn to scale representing the number of nucleotide substitutions per site.
Fig 4.
Rooted phylogenetic tree produced from Bayesian analysis using BEAST on 3777 base pairstructural proteins.
Analysis of32 taxa, model GTR+G+I of alphaviruses is shown. Posterior probabilities >0.7 are shown on major branches. Sequences of human Middelburg virus positives described in the current study are indicated with ZRU numbers and blue circles. Alphavirus complexes are indicated by brackets. The tree is drawn to scale representing the number of nucleotide substitutions per site.
Fig 5.
Rooted phylogenetic tree produced from Bayesian analysis using BEASTon non-structural and structural protein sequences.
Analysis of 36 taxa, model GTR+G+I of alphaviruses is shown. Probabilities >0.7 are shown on major branches. Sequences of human Middelburg virus positives described in the current study are indicated with ZRU numbers and blue circles. Alphavirus complexes are indicated by brackets. The tree is drawn to scale representing the number of nucleotide substitutions per site.
Fig 6.
Schematic representation of possible recombination events within structural regions of ZRU099/17 and ZRUH399/17.
Possible recombination events within the structural region) for ZRU099/17 and ZRUH399/17 are shown.Only detection models with significant statistical support (p<0.05) are shown. Different genes encoding for the structural protein are indicated with the recombination event indicated in the green block. Nucleotide position corresponds to that of ZRU099/17. SFV Semliki Forest Virus (Genbank accession number CAA27742); nt = nucleotide.