Genetic analysis of members of the species Oropouche virus and identification of a novel M segment sequence

Abstract

Oropouche virus (OROV) is a public health threat in South America, and in particular in northern Brazil, causing frequent outbreaks of febrile illness. Using a combination of deep sequencing and Sanger sequencing approaches, we determined the complete genome sequences of eight clinical isolates that were obtained from patient sera during an Oropouche fever outbreak in Amapa state, northern Brazil, in 2009. We also report the complete genome sequences of two OROV reassortants isolatd from two marmosets in Minas Gerais state, south-east Brazil, in 2012 that contained a novel M genome segment. Interestingly, all 10 isolates possessed a 947 nt S segment that lacked 11 residues in the S-segment 3' UTR compared with the recently redetermined Brazilian prototype OROV strain BeAn19991. OROV maybe circulating more widely in Brazil and in the non-human primate population than previously appreciated, and the identification of yet another reassortant highlights the importance of bunyavirus surveillance in South America.

Fig. 1.. Location of samples sequenced in this study. The map also shows Iquitos and Madre de Dios in Peru where OROV M segment reassortants were isolated, and Tucuruí, a municipality in Para, Brazil, where JATV was isolated. AC, Acre; AM, Amazonas; AP, Amapa; BA, Bahia; CE, Ceara; GO, Goias; MA, Maranhao; MG, Minas Gerais; MS, Mato Grosso do Sul; MT, Mato Grosso; PA, Para; PI, Piaui; PR, Parana; RO, Rondonia; RR, Roraima; SC, Santa Catarina; SP, Sao Paulo; RS, Rio Grande do Sul; TO, Tocantins.

Fig. 2.. Comparison of UTR sequences. (a) Comparison of the UTRs of BeH759022 isolate (chosen as a representative of the clinical isolates) with OROV strain BeAn19991. The bases in red highlight differences between BeAn19991 and BeH759022. (b) Minigenome assay. Comparison of S-segment-based minigenomes containing the S UTR of OROV BeAn19991 or the S UTR of the newly sequenced isolates. BSR-T7/5 cells were transfected with pTM1OROV-L and pTM1OROV-N plasmids expressing the L and N proteins, respectively, in addition to an S-segment-minigenome-expressing plasmid and pTM1-FF-Luc expressing firefly luciferase as an internal control. The control cells lacked pTM1OROV-L. Minigenome activity was expressed as fold induction over the background control. (c) Comparison of the M-segment UTRs of the novel M segment (BeAn790177) with those of OROV, Iquitos virus (IQTV), Madre de Dios virus (MDDV) and JATV. The C/A mismatch is highlighted in red. The dotted line indicates the extent of the conserved terminal sequence.

Fig. 3.. Phylogenetic trees of the Simbu serogroup viruses. The trees were recreated using a maximum-likelihood method based on the general time reversible model (GTR) with five rate categories and assuming sites are evolutionary invariable, for the L gene (a), the GTR model with discrete gamma distribution for the M polyprotein gene (b) and the Tamura three-parameter model with discrete gamma distribution for the N gene (c). Bars, number of nucleotide substitutions per site. Positions with lower than 95 % site coverage were eliminated. Alignment and analysis were conducted in mega6 (Tamura et al., 2013) and final trees were created using FigTree v.1.4.2.

Fig. 4.. Amino acid comparisons among viruses comprising the species Oropouche virus. (a) Pairwise amino acid p-distance scores of BeH759022 and BeAn790177 with Oropouche virus species and Utinga virus (UTIV). (b) M-segment deduced amino acid similarity plot using OROV as a query sequence and IQTV, MDDV, JATV and BeAn790177 as reference sequences.

Fig. 5.. Phylogenetic trees of viruses comprising members of the species Oropouche virus. (a) Maximum-likelihood phylogeny of the L gene with bootstrap support/Bayesian posterior probability shown on the branch. (b) Maximum-likelihood phylogeny of the M polyprotein gene with bootstrap support/Bayesian posterior probability shown on the branch. (c) Maximum-likelihood phylogeny of the N gene with bootstrap supports/Bayesian posterior probability shown on the branch. In (a)–(c), * represents 100 % bootstrap support. Isolates sequenced in this paper are highlighted in red. Full details of the strains used in this analysis are presented in Table S2. Bars, number of nucleotide substitutions per site. Clades A–D are indicated.

Fig. 6.. Reassortment among viruses comprising the species Oropouche virus. Maximum-likelihood phylogeny of the L segment with each isolate annotated with their clade assignment (A–D) according to the L-, M- and S-segment phylogenies. The different patterns represent the different interclade reassortments: pattern 1, C-D-D; pattern 2, C-B-D; pattern 3, C-A-D; pattern 4, D. Isolates sequenced in this paper are highlighted in red.