Human Orthobunyavirus Infections , Tefé , Amazonas , Brazil

Authors

results described in this study strengthen that this arbovirus, and its closely-related recombinants, should be under continuous surveillance, at least in the endemic countries of Latin America.
Between April and June 2015, professionals of the health surveillance system at Tefé (03° 21′ 14″ S; 64° 42′ 39″ W), a mid-size city located in the interior of the Amazonas state, Brazil, collected serum samples from a series of dengue suspected cases.The specimens were obtained between the first and second day after symptom onset and sent to Fundação de Medicina Tropical -Dr.Heitor Vieira Dourado (FMT-HVD) at Manaus, Amazonas, Brazil for dengue virus (DENV) testing using RT-PCR .
Thirty DENV negative samples were randomly selected and sent to Instituto Leônidas e Maria Deane (ILMD), Fiocruz Amazônia, to investigate other Amazonian endemic arboviruses .At ILMD, samples were tested using a multiplexed reverse transcription real-time PCR protocol for Oropouche, and Oropouche-like viruses, (OROV, Peribunyaviridae family, genus Orthobunyavirus), targeting the S segment; as well as for Mayaro virus (MAYV, Togaviridae family, Alphavirus genus) aiming the NSP1 coding region .All patients gave written informed consent, and the principal investigators anonymized the participants' data.The ethics committee of FMT-HVD approved the study under the registration number 700.915 We detected nine OROV or OROV-like positive cases (TF031; TF036; TF070; TF081; TF086; TF087; TF100; TF101; TF103), seven males and two females, with a mean age of 36.2 years (range 8 -63), living in either rural or urban areas of Tefé.Regarding the clinical symptoms, all nine patients reported fever and headache; two patients also reported myalgia and arthralgia and one showed retro-orbital pain.Therefore, these patients presented non-specific symptoms that could easily lead to dengue misdiagnosis, if no specific laboratory testing were applied.
To further confirm these results, all nine samples were submitted to an RT-PCR protocol targeting the S segment of OROV/OROV-like viruses using primers OROV_S_134F 5′-CGGACAAGTGCTCAATGCTG -3′ and OROV_S_734F 5′-GTCAATTCCGAATTGGCGCA -3′, generating a 601bp amplicon.These products were used for capillary nucleotide sequencing in an ABI3130 genetic analyzer, installed at the ILMD genomics platform.After removal of the primers, the final 558bp sequence for each sample was used for a multiple alignment with MAFFT , embedded in Geneious Software 10.2.2 .The orthobunyavirus sequences obtained in this study were highly conserved among them, as should be expected for any arbovirus samples collected from the same area, in a small-time frame.Only one nucleotide variation was observed in the sample TF86, a silent mutation (Transversion: T / A) in position 386 related to the OROV reference sequence for segment S NC_005777.
We also conducted a BLAST analysis over the consensus nucleotide sequence, using the MegaBLAST algorithm .Only orthobunyavirus sequences were retrieved among the first 100 hits with the higher score and lower E-values.

Fig. 1 :
Fig. 1: Maximum likelihood tree of the genomic S segment of orthobunyaviruses.All ICTV recognized orthobunyavirus species with available genomic data for the S segment were used for phylogenetic reconstruction, together with Tefé samples and other OROV-like viruses.The clade containing the Tefé samples, grouped with the species Oropouche orthobunyavirus, and the closely-related OROV-like viruses, is highlighted in yellow.Branches are colored according to the aLRT support.