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1、VETERINARY MICROBIOLOGY - RESEARCH PAPER Divergent coronaviruses detected in wild birds in Brazil, including a central park in So Paulo Carla M. Barbosa1 University of Vale do Rio dos Sinos (UNISINOS); University of Pernambuco (UFRPE); National Center for Research and Conservation of Wild Birds (CEM
2、AVE); Laboratory of Bird Ecology, Institute of Biosciences (UFMT); and Laboratory of Virology and Rickettsioses, Veterinary Hospital (UFMT). Samples were collected from wild birds after capture with mist nets, strategically placed between 30 and 250 cm of the ground, and by hand nets on an open fiel
3、d in public parks. The field works were made according to the permission of the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA) and National Center for Research and Conservation of Wild Birds (CEMAVE) developed and followed protocols approved by the Ethics Committee on Ani
4、mal Experimentation of theInstituteof Biomedical Sciences,Universityof SoPaulo. Sampling from oropharyngeal and cloacal swabs was obtained and placed together in vials containing VTM transport medium 16. All birds were then released after sampling. RNA extraction and reverse transcription Viral RNA
5、was extracted using the MagMax TM-96 RNA Isolation Kit (Ambion, Austin, TX, USA) according to the manufacturers instructions. The extracted RNA was eluted in 80 L of RNase-free water and stored at 80 C until processed. The cDNA was transcribed using the High- Capacity cDNA Archive Kit (Applied Biosy
6、stems, Foster City, CA, USA) following the manufacturers instructions. The RT reaction was performed by adding, to each reaction, 10LoftotalRNA, 2LofRTBuffer10X,2 L ofRandom Primers (50 ng/L) (Thermo Fisher Scientific, Waltham, MA, USA), 4.2 L of DEPC water, and 50 U of MultiScribe ReverseTranscript
7、ase(ThermoFisherScientific).TheRTstep conditions were 25 C for 10 min, 37 C for 120 min, and 85 C for 5 min, and cooling to 4 C. Coronavirus detection Polymerase chain reaction (PCR) followed by nested PCR were performed according to the protocol for the detection of avian CoVs developed by Chu et a
8、l. (2011) 17, which targets the RNA-dependent RNA polymerase (RdRp) gene in the ORF1 area of the viral genome. The resulting approx- imately 600-bp PCR fragment and 440-bp nested PCR frag- ment were separated by agarose gel 1.5% electrophoresis and viewed under UV light after staining with ethidium
9、bro- mide (25 g/100 mL). Purification and sequencing Samples were purified using the ExoSap-IT enzyme (GE Healthcare, Chalfont St. Giles, UK) and GeneJET Gel Extraction Kit (Thermo Fisher Scientific) according to the Braz J Microbiol Table 1Surveillance of Coronavirus in resident and wild birds in B
10、razil by molecular tests. Bold represents positive samples SiteDateScientific nameSample*No. pos./No. birds Ilha de Maraj, PAJul. 2006Cairina mochataC and O0/42 Gallus gallusC and O0/36 Meliagres gallopavoC and O0/15 Ajuruteua, PANov.2006Actitis maculariusC and O0/5 Calidris pusillaC and O0/2 Charad
11、rius semipalmatusC and O0/1 Dendrocygna autumnalisC and O0/18 Arenaria interpresC and O0/9 Dendrocygna viduataC and O0/56 Ilha de Canelas, PANov. 2006Actitis maculariusC and O0/10 Calidris canutusC and O0/1 Calidris pusillaC and O0/3 Calidris minutillaC and O0/1 Sterna hirundoC and O0/4 Larus sp.C a
12、nd O0/1 Nov. 2008Actitis maculariusC and O0/31 Arenaria interpresC and O0/22 Calidris albaC and O0/2 Limnodromus griseusC and O0/9 Tringa melanoleucaC and O0/1 Charadrius semipalmatusC and O0/1 Gelochelidon niloticaC and O0/1 Bahia de So Marco, MAApr. 2007Dendrocygna viduataC and O0/27 Netta erythro
13、phthalmaC and O0/2 Dendrocygna autumnalisC and O0/48 Gallinula chloropusC and O0/2 Amazonetta brasiliensisC and O0/14 Anas bahamensisC and O0/13 Meliagres gallopavoC and O0/1 Lagoa do Peixe National Park, RSNov. 2009Rynchops nigerC and O1/63 Calidris fuscicolisC and O1/63 Haematopus palliatusC and O
14、0/6 Himantopus melanurusC and O0/2 Pluvialis dominicaC and O0/7 Arenaria interpresC and O0/4 Calidris canutusC and O0/3 Calidris albaC and O1/6 Calidris pusillaC and O0/1 Nycticriphes semicollarisC and O0/1 Fulica leucopteraC and O0/1 Puffinus gravisC and O0/1 Puffinus puffinusC and O0/3 Tringa flav
15、ipesC and O0/2 Sterna hirundoC and O0/15 Phalacrocorax brasilianusC and O0/2 Vanellus chilensisC and O0/1 Elaenia mesoleucaC and O0/1 Pluvialis squatarolaC and O0/1 Tryngites subruficollisC and O0/7 Braz J Microbiol manufacturers instructions. The Sanger sequencing reaction was prepared using Big Dy
16、e Terminator v3.1 Cycle Sequencing Kit (Life Technologies, Foster City, CA, USA), followed by purification using BigDye X Terminator Purification Kit (Applied Biosystems), and analysis in an au- tomated ABI PRISM 3130XL DNA Sequencer (Applied Biosystems). Genetic characterization and phylogenetic tree of the RdRp gene Generated chromatograms containing nucleotide sequences were visualized using Bioedit Sequence Alignment Editor (v.7.1.5.0). Nucleotide sequences were aligned using CLC sequence
