Oropouche virus detection in saliva and urine

Oropouche virus (OROV) is an arthropod-borne virus of the Peribunyaviridae family, transmitted to humans primarily by Culicoides paraensis. It is one of the main arboviruses infecting humans in Brazil, primarily in the Amazon Region. Here, we report the detection of OROV in the saliva and urine of a patient whose samples were collected five days after the onset of symptoms. Nucleotide sequencing and phylogenetic analysis further confirmed the results. To our knowledge, this is the first study reporting the detection of OROV in the saliva and urine of an infected patient. In addition, the results of our study expand the current knowledge pertaining to the natural history of Oropouche fever.

Key words:
arboviruses; orthobunyavirus; Oropouche virus; saliva; urine; real-time polymerase chain reaction

Authorship

Valdinete Alves do Nascimento

Fundação Oswaldo Cruz-Fiocruz, Instituto Leônidas e Maria Deane, Manaus, AM, BrasilFundação Oswaldo Cruz-FiocruzBrazilManaus, AM, BrazilFundação Oswaldo Cruz-Fiocruz, Instituto Leônidas e Maria Deane, Manaus, AM, Brasil

Fundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Programa de Pós-Graduação em Biologia Celular e Molecular, Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz-FiocruzBrazilRio de Janeiro, RJ, BrazilFundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Programa de Pós-Graduação em Biologia Celular e Molecular, Rio de Janeiro, RJ, Brasil

João Hugo Abdalla Santos

Universidade Federal do Amazonas, Manaus, AM, BrasilUniversidade Federal do AmazonasBrazilManaus, AM, BrazilUniversidade Federal do Amazonas, Manaus, AM, Brasil

Hospital Adventista de Manaus, Manaus, AM, BrasilHospital Adventista de ManausBrasilManaus, AM, BrasilHospital Adventista de Manaus, Manaus, AM, Brasil

Dana Cristina da Silva Monteiro

Karina Pinheiro Pessoa

Fundação Oswaldo Cruz-Fiocruz, Instituto Leônidas e Maria Deane, Programa de Pós-Graduação em Biologia da Interação Patógeno-Hospedeiro, Manaus, AM, BrasilFundação Oswaldo Cruz-FiocruzBrazilManaus, AM, BrazilFundação Oswaldo Cruz-Fiocruz, Instituto Leônidas e Maria Deane, Programa de Pós-Graduação em Biologia da Interação Patógeno-Hospedeiro, Manaus, AM, Brasil

Antonio José Leão Cardoso

Victor Costa de Souza

Ligia Fernandes Abdalla

Universidade do Estado do Amazonas, Manaus, AM, BrasilUniversidade do Estado do AmazonasBrazilManaus, AM, BrazilUniversidade do Estado do Amazonas, Manaus, AM, Brasil

Felipe Gomes Naveca ⁺+ Corresponding author: felipe.naveca@fiocruz.br

http://orcid.org/0000-0002-2888-1060

+ Corresponding author: felipe.naveca@fiocruz.br

AUTHORS’ CONTRIBUTION

VAN, LFA, JHAS, and FGN conceived the study; VAN, LFA, JHAS and FGN designed the study protocol; VAN, DCSM, KPP, AJLC, VCS and FGN performed the molecular tests and the analysis and interpretation of the data; LFA and JHAS collected clinical information; VAN and FGN wrote the manuscript; FGN financed the study; VAN, LFA and FGN critically revised the manuscript for intellectual content. All authors read and approved the final manuscript.

SCIMAGO INSTITUTIONS RANKINGS

Universidade Federal do Amazonas, Manaus, AM, BrasilUniversidade Federal do AmazonasBrazilManaus, AM, BrazilUniversidade Federal do Amazonas, Manaus, AM, Brasil

Hospital Adventista de Manaus, Manaus, AM, BrasilHospital Adventista de ManausBrasilManaus, AM, BrasilHospital Adventista de Manaus, Manaus, AM, Brasil

Universidade do Estado do Amazonas, Manaus, AM, BrasilUniversidade do Estado do AmazonasBrazilManaus, AM, BrazilUniversidade do Estado do Amazonas, Manaus, AM, Brasil

Figures | Tables

Figures (1)
Tables (1)

Thumbnail

Phylogenetic tree of Orthobunyavirus species. Three Bayesian trees, one for each genome segment, were constructed with MrBayes software v3.2.6 and 76 taxa (the 75 orthobunyavirus species recognised by the International Committee on Taxonomy of Viruses (ICTV) with complete genome records available in GenBank on 01-Jun-2019 and the sample BR_AM_ILMD_0240AOS_2016 reported in this study). Phylogenetic trees were set mid-rooted, with increased node order in FigTree 1.4.4 for clarity. A colour-key represents the posterior probability values of each branch. The clade containing the sequence described in this study is highlighted in yellow, clustered with the Oropouche virus RefSeq. The scale bar represents nucleotide substitutions per site. A: L segment tree; B: M segment tree; C: S segment tree.

Thumbnail

TABLE
Oligonucleotides designed and used in this study

Phylogenetic tree of Orthobunyavirus species. Three Bayesian trees, one for each genome segment, were constructed with MrBayes software v3.2.6 and 76 taxa (the 75 orthobunyavirus species recognised by the International Committee on Taxonomy of Viruses (ICTV) with complete genome records available in GenBank on 01-Jun-2019 and the sample BR_AM_ILMD_0240AOS_2016 reported in this study). Phylogenetic trees were set mid-rooted, with increased node order in FigTree 1.4.4 for clarity. A colour-key represents the posterior probability values of each branch. The clade containing the sequence described in this study is highlighted in yellow, clustered with the Oropouche virus RefSeq. The scale bar represents nucleotide substitutions per site. A: L segment tree; B: M segment tree; C: S segment tree.

TABLE Oligonucleotides designed and used in this study

Oligo	Sequence (5’- 3’)	Start	Stop
OROV_L_56_FNF	TTGCTCAACCARTATCGRAATAGGAT	56	81
OROV_L_174_FNF	CTGCAAAYCTTGAGTAYAGAAATGATG	174	200
OROV_L_621_FNR	TCAATCCATGGCAATGTCATTGT	621	599
OROV_M_2185_FNF	TCCCAAATCTAATCCTTTTACYGAT	2185	2209
OROV_M_2864_FNF	AGTATAGATGTACAAGGTACAGAATC	2864	2889
OROV_M_3564_FNR	TTCCTTCTCATAGCATGGCAT	3564	3544
OROV_S_6_FNF	TGTACTCCACAATTCAAAACAT	6	27
OROV_S_133_FNF	ACGGACAAGTGCTCAATGCT	133	152
OROV_S_728_FNR	TCCGAATTGGCGCAAGAAGT	728	709
Assay	Primer pairs		Size (bp)
L - 1^st PCR (55ºC)	OROV_L_56_FNF + OROV_L_621_FNR		566
L - semi-nested (50ºC)	OROV_L_174_FNF + OROV_L_621_FNR		448
M - 1^st PCR (55ºC)	OROV_M_2185_FNF + OROV_M_3564_FNR		1380
M - semi-nested (55ºC)	OROV_M_2864_FNF + OROV_M_3564_FNR		701
S - 1^st PCR (58ºC)	OROV_S_6_FNF + OROV_S_728_FNR		723
S - semi-nested (58ºC)	OROV_S_133_FNF + OROV_S_728_FNR		596

Start/Stop positions refers to the nucleotide position of OROV GenBank reference sequence NC_005776.1 (segment L), NC_005775.1 (segment M), and NC_005777.1 (segment S). To increase sensitivity, we developed semi-nested reactions for each genome segment. All 1st polymerase chain reaction (PCR) reactions followed the same program: 94ºC for 2 min for enzyme activation; 35 cycles (94ºC for 30 s, 55 or 58ºC for 30 s, and 72ºC during 1 min/Kb), a final step at 72ºC for 5 min. For semi-nested reactions: 94ºC for 2 min for enzyme activation; 30 cycles (94ºC for 30 s, 50, 55 or 58ºC for 30 s, and 72ºC for 1 min), a final step at 72ºC for 5 min. All primers used in this study were synthesised by IDT DNA Technology, USA.

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Oropouche virus detection in saliva and urine

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[1] + Corresponding author: felipe.naveca@fiocruz.br