Jpn. J. Infect. Dis., 73, 164–165, 2020

Short Communication Oropouche Detection in Febrile Patients’ Saliva and Urine Samples in Salvador, Bahia, Brazil Larissa Moraes dos Santos Fonseca1*, Rejane Hughes Carvalho1, Antonio Carlos Bandeira2, Silvia Ines Sardi1, and Gubio Soares Campos1 1Laboratory of , Health Science Institute, Federal University of Bahia, Bahia; and 2Faculty of Technology and Sciences Medical School, Bahia, Brazil

SUMMARY: Oropouche virus (OROV) is a negative-sense, single-stranded RNA transmitted to humans by the midge Culicoides paraenesis, causing Oropouche fever. Reports of its outbreak in Brazil have so far been restricted to the Central-Northern region of the country. However, its incidence is underestimated, mainly due to its clinical similarities with other arbovirus diseases, including dengue (DENV), (CHIKV), and zika (ZIKV), and the lack of specific diagnostic tests. Here, we report for the first time, the detection of OROV in saliva and urine samples, and cases of autochthone OROV in Salvador Metropolitan region, Bahia, a Northeastern capital in the coast of Brazil. Serum, saliva, and urine samples negative for DENV, CHIKV, and ZIKV were tested for OROV using a reverse transcription nested polymerase chain reaction (RT-nested-PCR) protocol, and 2 serum, 2 saliva, and 1 urine samples were positive. This report shows the need for an efficient surveillance system for controlling the spread of this virus, and suggests the use of saliva and urine as alternative samples for OROV detection in the absence of serum samples.

The oropouche virus (OROV) is an arbovirus in region, and sporadic cases from the Central and the genus , of the Peribunyaviridae Southeast regions of the country (7). family, transmitted to humans by the midge Culicoides The study protocol was approved by the Conselho paraenesis (1). OROV causes Oropouche fever and Nacional de Ética em Pesquisa [National 140 Ethics has an ranging from 4 to 8 days, Committee] (protocol 45483115.9.0000.0046). All with clinical manifestations such as acute high fever, participants provided written informed consent. headache, muscle and joint pain, and (2). In this study, we detected OROV in serum, saliva, The disease is either undiagnosed or misdiagnosed, and urine samples of patients presenting with arbovirus due to its mild, self-limiting manifestations, symptom symptoms, who tested negative for DENV, similarity to other endemic arboviral diseases in Brazil CHIKV, and ZIKV. It is also the first time OROV was such as dengue (DENV), chikungunya (CHIKV), and detected in the Metropolitan region of Salvador, Bahia, zika (ZIKAV) virus infections, and the lack of molecular which occupies the Northeast region of Brazil. differential diagnostic methods in endemic regions (3). Serum (n = 53), saliva (n = 42), and urine (n = 22) Like all , OROV contains a samples were collected from 53 patients with dengue- tripartite, single-stranded, negative-sense RNA genome like symptoms, who reported to a private hospital in the named by size: large (L), medium (M), and Small city of Salvador, Bahia, Brazil, from November/2016 (S). The L segment encodes the viral polymerase; M to December/2017. Serum was obtained from all segment, glycoproteins Gn and Gc, and a nonstructural the patients, but only 42 and 22 had saliva and urine protein NSm; and S segment, nucleocapsid protein samples, respectively. (N) and a second non-structural protein (NSs) from All patients tested negative for CHIKV, DENV, and overlapping reading frames (4). ZIKV. Using the commercial serology test Dengue Ns1/ Since its first human infection in Trinidad and Tobago IgG/IgM ECO Teste (ECO Diagnóstica, Lima, Brazil, in 1955 (5), OROV has been detected in Bolívia, Corinto-MG), the serum samples were tested for DENV, Brazil, Argentina, Panama, and Peru (2,4). In Brazil, in the hospital. CHIKV and ZIKV were screened via the virus was first isolated in 1960 in Belém-Pará, from molecular diagnosis, as described by Edwards et al. (8) a sylvestre animal, the pale-throated (Bradypus and Lanciotti et al. (9), respectively, in our laboratory. tridactylus) (6). In subsequent years, human infections OROV was detected using a RT-nested-PCR targeting have been reported in the urban areas of the Northern the S RNA segment, as described by Morelli et al. (10). Viral RNA was extracted from the samples using the Received August 2, 2019. Accepted October 24, 2019. QIAmp viral RNA kit (QIAGEN, Hilden, Germany). The J-STAGE Advance Publication November 29, 2019. outer primers BUN-S (5-AGTAGTGTGCTCCAC-3) DOI: 10.7883/yoken.JJID.2019.296 and BUN-C (5-AGTAGTATACTCCAC-3), were used *Corresponding author: Mailing address: Av. Reitor Miguel for the first RT-PCR round. They span the 3 genome Calmon, s/n, Salvador, Bahia, Brazil, 40110-902, Brazil. segment extremities in the Peribunyaviridae family, Tel: +55 71 98237-6909, Fax: +55 71 3283-8894, E-mail: amplifying a 700 bp outer product. The inner primers [email protected] BS-S (5- GTGGGGTCCAATTTGC-3) and BS-C

164 Oropouche Virus in Saliva and Urine Samples

(5-TGAACCCTATGCATCT-3), were used for the method, since it is easy, non-invasive (particularly second PCR round, generating a 300 bp product. The beneficial for diagnosing children), and cheap to inner PCR products were analysed via electrophoresis transport. on a 2% ethidium bromide-stained agarose gel. Finally, Oropouche fever’s neglect in Brazil may The second-round PCR products of positive samples be owed to the similarity of its symptoms with those were purified with the QIAmp PCR purification kit of common diseases caused by other . (QIAGEN) and sequenced in both directions, using the Through this report, we alert the Brazilian public health inner primers and Big Dye Terminator chemistry V3.1 authorities and recommend improved methods for (Applied Biosystems, Foster City, CA, USA), in an ABI- differential arbovirus diagnosis. If OROV infection is Prism 3500 Genetic Analyzer (Applied Biosystems). not diagnosed, it may lead to periodic outbreaks like in Five individual patient samples (2 sera, 2 saliva, and DENV, CHIKV in 2014, or ZIKV in 2015 (14,15). 1 urine) were positive for OROV. BLAST analysis of the OROV-positive nested-PCR amplicon sequences Acknowledgments This work was funded by the Zika fast track showed 97 to 99% identity with the reference sequence – CAPES project n° 88887-116628/2016. The funding has been corresponding to the S RNA segment encoding the granted to the author Gubio Soares Campos. We thank Daniele nucleocapsid protein (N) and non-structural protein Freitas Henriques, from the Evandro Chagas Institute, Belém-PA, (NSs). The sequences were submitted to GenBank for providing us with control samples of the Oropouche virus. (Accession numbers: MF155931, MK875765-68, Conflict of interest None to declare. respectively). This is the first report of OROV infection in the REFERENCES Metropolitan Region of Bahia State, although it has been reported in nearby regions (2). Since Bahia State is 1. Pinheiro FP, Travassos da Rosa AP, Gomes ML. Transmission of geographically distant from the official OROV-endemic Oropouche virus from man to hamster by the midge Culicoides paraensis. Science. 1982;215:1251-3. areas, the virus has probably been spread across the 2. Sakkas H, Bozidis P, Franks A, et al. Oropouche fever: a review. country via intense human transit. . 2018;10:pii:E175. OROV outbreaks are concentrated in Northern 3. Lopes N, Nozawa C, Linhares RE. General features and Brazilian cities. However, sporadic cases have been epidemiology of emerging arboviruses in Brazil. Rev Pan-Amaz reported in the Central, Northeast, and Southeast regions Saude. 2014;5:55-64. Portuguese. 4. Travassos da Rosa JF, de Souza WM, Pinheiro FP, et al. Oropouche (7). Furthermore, OROV has been isolated from different virus: clinical, epidemiological, and molecular aspects of a non-human hosts (Bradypus tridactulus, Callithrix sp., neglected orthobunyavirus. Am J Trop Med Hyg. 2017;96:1019-30. and wild birds) and invertebrate vectors (C. paraensis, 5. Anderson CR, Spence L, Downs WG, et al. Oropouche virus: a new , Coquillettidia venezuelensis, human disease agent from Trinidad, West Indies. Am J Trop Med Hyg. 1961;10:574-8. and Ochlerotatus serratus) (4), which can contribute to 6. Pinheiro F, Pinheiro M, Bensabath G, et al. Oropouche vírus the spread of the virus throughout the country, including in Belém. Rev Serv Sau Pub. 1962; 12:15-23. Portuguese. Bahia State. 7. Nunes MRT, Martins LC, Rodrigues SG, et al. Oropouche virus None of the OROV positive subjects in this report isolation, Southeast Brazil. Emerg Infect Dis. 2005;11:1610-3. visited the documented OROV circulating areas in the 8. Edwards CJ, Welch SR, Chamberlain J, et al. Molecular diagnosis and analysis of Chikungunya virus. J Clin Virol. 2007;39:271-5. month preceding symptom onset, providing evidence for 9. Lanciotti RS, Kosoy OL, Laven JJ, et al. Genetic and serologic the autochthonous circulation of the virus in Bahia State. properties of associated with an , Yap State, Interestingly, to the best of our knowledge, the use Micronesia. Emerg Infect Dis. 2008;14:1232-9. of saliva and urine samples for OROV investigation, 10. Moreli ML, Aquino VH, Cruz AC, et al. Diagnosis of oropouche had not been reported. Detection of viruses in saliva virus infection by RT-nested-PCR. J Med Virol. 2002;66:139-42. 11. Andries AC, Duong V, Ly S, et al. Value of routine dengue and urine samples could be associated with persistent diagnostic tests in urine and saliva specimens. Plos Negl Trop Dis. viral load after acute phase disease (11). In Dengue-like 2015; 9: e0004100. illness, the patient reports to the hospital after several 12. Hirayama T, Mizuno Y, Takeshita N, et al. Detection of dengue days of fever, which could interfere with virus detection virus genome in urine by real-time reverse transcriptase PCR: a laboratory diagnostic method useful after disappearance of the in during the acute phase (12). In such cases, genome in serum. J Clin Microbiol. 2012;50:2047-52. saliva and urine could be excellent alternative samples. 13. Gourinat AC, O’Connor O, Calvez E, et al. Detection of Zika virus Several reports have demonstrated viral infection in urine. Emerg Infect Dis. 2015;21:84-6. diagnosis using saliva and urine samples, including the 14. Nunes MR, Faria NR, de Vasconcelos JM, et al. Emergence and detection of other arboviruses (11–13). Some studies potential for spread of Chikungunya virus in Brazil. BMC Med. 2015;13:102. also revealed that urine could support higher viral loads 15. Campos GS, Bandeira AC, Sardi SI. Zika virus outbreak, Bahia, than blood, and for a longer period (12). Moreover, Brazil. Emerg Infect Dis. 2015;21:1885-6. diagnosis using saliva and urine could be an effective

165