Divergent Coronaviruses Detected in Wild Birds in Brazil, Including a Central Park in São Paulo
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Brazilian Journal of Microbiology (2019) 50:547–556 https://doi.org/10.1007/s42770-019-00065-7 VETERINARY MICROBIOLOGY - RESEARCH PAPER Divergent coronaviruses detected in wild birds in Brazil, including a central park in São Paulo Carla M. Barbosa1 & Edison L. Durigon1 & Luciano M. Thomazelli1 & Tatiana Ometto1 & Roberta Marcatti2 & Marcello Shiavo Nardi2 & Daniel M. de Aguiar3 & João Batista Pinho4 & Maria Virginia Petry5 & Isaac Simão Neto6 & Patrícia Serafini7 & Roberta Costa Rodrigues8 & Severino Mendes de Azevedo Junior9 & Luiz Gustavo B. Góes1 & Jansen de Araujo1 Received: 11 August 2018 /Accepted: 7 December 2018 /Published online: 15 March 2019 # Sociedade Brasileira de Microbiologia 2019 Abstract Coronaviruses are single-stranded positive-sense RNA viruses associated with important avian diseases. Their relatively high rates of mutation and recombination frequencies allow them to adapt to new hosts and ecological niches. Although Brazil has 18% of global avian species diversity, studies regarding the presence of avian viral diseases in wild birds in South America are scarce. In this study, we performed a retrospective analysis of the presence of CoVs in 746 wild birds. Oropharyngeal and cloacal swabs were obtained and placed together in vials containing VTM transport medium collected in different regions of Brazil between 2006 and 2013. Screening for viral nucleic acid was performed using conventional RT-PCR and pancoronavirus nested PCR. Positive samples were character- ized by partial sequencing of the RNA-dependent RNA polymerase (RdRp) gene, and ensuing phylogenetic analysis was performed to investigate the association between virus epidemiology and bird migration routes. Coronavirus RNA were detected and sequenced from six samples, in which three were related to gammacoronaviruses group and the other three to deltacoronavirus group. Our study documents the presence of CoVs related to avian gamma- and deltacoronaviruses circulating in both urban- and poultry-farm regions of Brazil, implicating wild birds as potential carriers of CoVs which may represent a risk to poultry farms and public health in Brazil. Keywords Gammacoronavirus . Deltacoronavirus . Wild birds . Brazil . South America Introduction Infectious bronchitis virus (IBV), known as coronavirus of chickens, causes important economic losses to the poultry in- Coronaviruses are RNA single-stranded positive polarity virus dustry. Studies about the presence of CoVs in wild birds, es- associated with important disease in avian poultry [1]. pecially IBV, in South America are scarce [2]. Coronaviruses Responsible Editor: Fernando Spilki Electronic supplementary material The online version of this article (https://doi.org/10.1007/s42770-019-00065-7) contains supplementary material, which is available to authorized users. * Jansen de Araujo 5 Universidade do Vale do Rio dos Sinos (UNISINOS), São [email protected] Leopoldo, Rio Grande do Sul, Brazil 6 Instituto Chico Mendes de Conservação da Biodiversidade 1 Institute of Biomedical Sciences, University of Sao Paulo, 1374 Prof. (ICMBio), Brasília, Brazil Lineu Prestes avenue, São Paulo 05508-900, Brazil 7 2 Wildlife and Green Areas Department, São Paulo Municipality, São Centro Nacional de Pesquisa e Conservação de Aves Silvestres Paulo, Brazil (CEMAVE), Cabedelo, Paraíba, Brazil 8 3 Laboratório de Virologia e Rickettsioses, Hospital Veterinário, Universidade Federal da Paraíba (UFPB), João Pessoa, Paraíba, Universidade Federal de Mato Grosso, Cuiabá, Mato Grosso, Brazil Brazil 4 Laboratório de Ecologia de Aves, Instituto de Biociências, 9 Universidade Federal Rural de Pernambuco (UFRPE), Universidade Federal de Mato Grosso, Cuiabá, Mato Grosso, Brazil Recife, Pernambuco, Brazil 548 Braz J Microbiol (2019) 50:547–556 (CoVs) possess the largest genomes among RNA viruses and of the Wildlife Department of São Paulo (DEPAVE); University have relatively high mutation rates and recombination frequen- of Vale do Rio dos Sinos (UNISINOS); University of cies when compared with other single-strand RNA viruses, Pernambuco (UFRPE); National Center for Research and which may allow them to adapt to new hosts and ecological Conservation of Wild Birds (CEMAVE); Laboratory of Bird niches [3–5]. These characteristics are exemplified by the two Ecology, Institute of Biosciences (UFMT); and Laboratory of emerged highly pathogenic betacoronaviruses, CoV-SARS Virology and Rickettsioses, Veterinary Hospital (UFMT). (severe acute respiratory syndrome) and the CoV-MERS Samples were collected from wild birds after capture with mist (Middle East respiratory syndrome) [6]. nets, strategically placed between 30 and 250 cm of the ground, Even though avian coronaviruses with zoonotic potential have and by hand nets on an open field in public parks. The field not been reported yet, many species of wild and domestic birds works were made according to the permission of the Brazilian can carry CoVs responsible for substantial economic loss within Institute of Environment and Renewable Natural Resources the poultry industry such as the gammacoronavirus infectious (IBAMA) and National Center for Research and Conservation bronchitis virus (IBV). This virus affects the gastrointestinal, of Wild Birds (CEMAVE) developed and followed protocols respiratory, urinary, and reproductive systems and may lead to approved by the Ethics Committee on Animal Experimentation mortality rates as high as 30% and sudden drops in egg produc- of the Institute of Biomedical Sciences, University of São Paulo. tion [7]. Moreover, many novel CoVs genetically distinct from Sampling from oropharyngeal and cloacal swabs was obtained IBV have been recently identified in different families of wild and placed together in vials containing VTM transport medium and domestic birds and mammals but their potential economic [16]. All birds were then released after sampling. losses remain unknown. Recently, Durães-Carvalho et al. (2015), described the detection of delta- and betacoronaviruses in captive RNA extraction and reverse transcription wild birds, from a rehabilitation center, originally captured in São Paulo and Mato Grosso do Sul state with close evolutionary Viral RNA was extracted using the MagMax TM-96 RNA relationships with CoVs isolated from mammals [8]. Isolation Kit (Ambion, Austin, TX, USA) according to the Wild migratory birds may facilitate the spread of viruses, as manufacturer’s instructions. The extracted RNA was eluted demonstrated by the surveillance of avian influenza virus [9, in 80 μL of RNase-free water and stored at − 80 °C until 10] and West Nile virus [11–13], but the circulation of CoV in processed. The cDNA was transcribed using the High- avian migratory routes in South America, more specifically in Capacity cDNA Archive Kit (Applied Biosystems, Foster Brazil, still need to be elucidated. The potential diversity of City, CA, USA) following the manufacturer’s instructions. CoV in Brazilian bird fauna as consequence of the bird species The RT reaction was performed by adding, to each reaction, diversity (18%), together with the large number of endangered 10 μL of total RNA, 2 μLofRTBuffer10X,2μL of Random species and the high intensity of poultry farms in Brazil, indi- Primers (50 ng/μL) (Thermo Fisher Scientific, Waltham, MA, cates that birds from Brazilian biomes are important subjects USA), 4.2 μL of DEPC water, and 50 U of MultiScribe™ for CoV surveillance [2, 14]. Reverse Transcriptase (Thermo Fisher Scientific). The RTstep In this study, we performed a retrospective analysis, mo- conditions were 25 °C for 10 min, 37 °C for 120 min, and lecular detection, and phylogenetic analysis of avian 85 °C for 5 min, and cooling to 4 °C. coronaviruses using oropharyngeal and cloacal swab samples from wild and domestic birds collected between 2006 and Coronavirus detection 2013 in different regions of Brazil to investigate the associa- tion between virus epidemiology, bird migration routes, and Polymerase chain reaction (PCR) followed by nested PCR the proximity of urban areas and poultry farms. were performed according to the protocol for the detection of avian CoVs developed by Chu et al. (2011) [17], which targets the RNA-dependent RNA polymerase (RdRp) gene Materials and methods in the ORF1 area of the viral genome. The resulting approx- imately 600-bp PCR fragment and 440-bp nested PCR frag- Sample collection ment were separated by agarose gel [1.5%] electrophoresis and viewed under UV light after staining with ethidium bro- In total, 746 samples (Table 1) of birds captured between 2006 mide (25 μg/100 mL). and 2013 in five expeditions to north, northeast, midwest, south, and southeast Brazil (Fig. 1) were analyzed. For selection, the Purification and sequencing migratory routes were investigated through a map analysis and experience of ornithologists [15]. The samples were collected by Samples were purified using the ExoSap-IT enzyme (GE field team of the Laboratory Clinical and Molecular Virology, Healthcare, Chalfont St. Giles, UK) and GeneJET™ Gel University of São Paulo (USP), São Paulo, Brazil, with support Extraction Kit (Thermo Fisher Scientific) according to the Braz J Microbiol (2019) 50:547–556 549 Table 1 Surveillance of Coronavirus in resident and wild birds in Brazil by molecular tests. Bold represents positive samples Site Date Scientific name Sample* No. pos./No. birds Ilha de Marajó, PA Jul. 2006 Cairina mochata CandO 0/42 Gallus gallus CandO 0/36 Meliagres gallopavo CandO 0/15 Ajuruteua, PA Nov.2006 Actitis macularius CandO 0/5 Calidris