Coronaviruses Detected in Brazilian Wild Birds Reveal Close Evolutionary Relationships with Beta- and Deltacoronaviruses Isolated From Mammals.

This study showed that the most of the coronaviruses (CoVs) detected in Brazilian wild birds clustered with the mouse hepatitis virus A59 strain, belonging to the BetaCoV group. Furthermore, CoV detected in two different bird species, Amazona vinacea and Brotogeris tirica, clustered with a CoV isolated from Sparrow (SpaCoV HKU17) belonging to a monophyletic group related with the CoVs isolated from swines (PorCoV HKU15), both belonging to the DeltaCoV genus, previously unreported in South America. Considering the risk of inter-species host switching and further adaptation to new hosts, detection in bird species of CoVs closely related to mammal CoVs should warn for the potential emergence of new threatening viruses.

Coronaviruses (CoVs), belonging to the order Nidovirales, family Coronaviridae, and subfamily Coronavirinae, are grouped into the AlphaCoV, BetaCoV, GammaCoV, and DeltaCoV genera (Woo et al. 2011). Alpha- and BetaCoVs infect or cause disease in mammals, while GammaCoV are almost exclusively found in birds, except for the CoV detected in beluga whale and bottlenose dolphin (Mihindukulasuriya et al. 2008; Woo et al. 2010). The fourth genus (DeltaCoV) was described recently and has already been identified in pigs and birds (Woo et al. 2011).

In this research, 368 cloacal and tracheal swabs from 33 bird species were collected in different regions belonging to the States of São Paulo (Bosque dos Jequitibás, Campinas-SP; Rio Claro-SP; Rehabilitation center [CRAS] Parque Ecológico do Tietê, São Paulo-SP) and Mato Grosso do Sul (CRAS Campo Grande-MS), Brazil. The viral RNA was extracted (QIAmp Viral RNA Mini kit, QIAGEN, Hilden, Germany), and the cDNA synthesis was performed following the manufacturer’s protocol (Applied Biosystems, Foster City, USA).

A PanCoV PCR selective essay was performed targeting a 440 bp of RNA-dependent RNA polymerase (RdRp) gene (Chu et al. 2011). Nineteen samples (5.2 %) distributed in 11 bird species were positive for CoV. The positive CoV species are as follows: Amazona vinacea, Ara ararauna, Asio clamator, Brotogeris tirica, Colaptes campestris, Columba livia, Coragyps atratus, Megascops choliba, Pitangus sulphuratus, Pyroderus scutatus, and Rupornis magnirostris (See Supplementary Table S1).

Different CoV sequences were used to represent the four CoV genera of the family Coronaviridae. Phylogenetic inference was reconstructed and implemented in FastTree 2.1.7 software (Price et al. 2010) using the Maximum-Likelihood (ML) method with the General Time-Reversible (GTR) model of nucleotide evolution with 20 rates of gamma distribution and 1.000 Shimodaira–Hasegawa-like (SH-like) values.

Our results showed that most of the samples clustered with the mouse hepatitis virus A59 strain (MHV A59) belonging to the BetaCoV group. Mice are the natural hosts of MHV A59, which is associated to hepatotropic and neurotropic diseases (Eriksson et al. 2008). Besides, two of our samples that were, respectively, detected in A. vinacea (Parrot-breasted-purple) and B. tirica (Plain Parakeet) clustered with CoV isolated from Sparrow (SpaCoV HKU17) belonging to a monophyletic group related with the CoVs isolated from swines (PorCoV HKU15), both belonging to the DeltaCoV genus (Fig. 1), previously unreported in South America. PorCoV HKU15 was detected in April 2014 in the USA (Wang et al. 2014), in 2015, which was directly associated to play a key role in diarrheic disease in swine; no treatment or vaccines are currently available (Hu et al. 2015; Ma et al. 2015).

Fig. 1

Molecular phylogenetic inference with Alpha-, Beta-, Gamma-, and DeltaCoV groups by ML method under GTR evolutionary model for the partial RdRp gene (267 bp). The colors represent CoVs detected in wild birds grouped with BetaCoV (red) and DeltaCoV (highlighted in blue) groups. The asterisk represents the bird samples from which the DNA sequences were obtained. The number within parenthesis shows the nucleotide identities of PorCoV HKU15 and SpaCoV HKU17 (both DeltaCoVs) with the CoV detected in Amazona vinacea. The letter X represents the arithmetic mean of nucleotide identities between the CoVs detected in the study and the MHV A59. The GenBank accession numbers of sequences deposited (from study) and retrieved from the database are shown in the tree. PorCoV porcine coronavirus; HCoV human coronavirus; PEDV porcine epidemic diarrhea virus; BatCoV bat coronavirus; TGEV transmissible gastroenteritis coronavirus; CCoV canine coronavirus; IBV infectious bronchitis virus; TCoV turkey coronavirus; MHV mouse hepatitis virus; BCoV bovine coronavirus; SARS severe acute respiratory syndrome; MERS middle east respiratory syndrome coronavirus; PToV (outgroup) porcine torovirus. The numbers above the branches represent the SH-like support values of 1000 replicates. Only values greater than or equal to 70 % are presented. Online version in color

Finally, we highlighted in this study the evolutionary proximity of CoVs detected in wild birds with Beta- and DeltaCoVs, both isolated from mammal and involved in the pathogenicity of their hosts. We believe that these findings may be useful for the understanding of CoVs’ evolutionary dynamics. Considering the risk of inter-species host switching and further adaptation to new hosts, detection in bird species of CoVs closely related to mammal CoVs should warn for the potential emergence of new threatening viruses.

Below is the link to the electronic supplementary material.

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