Genetically Diverse Coronaviruses in Captive Bird Populations in a Brazilian Zoological Park

Introduction

T he outbreaks of highly pathogenic avian influenza viruses around the globe in the past few years have been devastating for the countries concerned and have created fear in the general population. Similarly, in 2002 the world witnessed the emergence of severe acute respiratory syndrome in Guangdong Province, China, caused by a previously unknown coronavirus (CoV) (Liu 2003). It is likely that additional, yet to be identified CoVs are circulating in domestic birds, especially in tropical countries. The three existing groups of CoVs were initially distinguished based on the lack of an antigenic relationship between them (Masters 2006). Group 3 CoVs, which are genetically similar to infectious bronchitis virus (IBV), have been detected in healthy galliform and nongalliform birds (Cavanagh et al. 2002). Recently, populations of wild birds in northern England were found to have IBV-like RNA in their feces (Hughes et al. 2009). Currently, little information is available about the role of South American bird populations as reservoirs or long-distance vectors of CoV-like viruses.

Viral diseases can cause substantial mortality in both wild and captive bird populations. Brazil is a country with many migrating wild birds, which is likely to facilitate disease transmission from migratory to nonmigratory flocks in zoological parks or on farms. As viral detection and identification techniques become increasingly sophisticated, and as the study of wildlife diseases increases, it is likely that new viruses will be discovered and new diseases will be encountered (Hughes et al. 2009). Therefore, studies of the viral diseases of wild and captive birds are necessary. The latest techniques of molecular epidemiology can help scientists understand how viruses move from species to species and from region to region (Hughes et al. 2009, Jackwood et al. 2010).

The present study demonstrates the molecular detection and characterization of viruses from captive exotic bird populations sampled in São Paulo State Zoological Park in Brazil, which appear to be related to group 3 CoVs.

Materials and Methods

Results and Discussion

Captive animals in zoos are part of a complex ecosystem that includes free-roaming animals and their associated infectious diseases, including respiratory viruses. These results, obtained from captive birds in Brazil, represent the first report of a circulating CoV inside a zoological park. The CoV RNA was detected in eight individual fecal samples (Table 1), which corresponds to an individual prevalence estimate of 1.12% (95% confidence interval 0.5–2.7). Of the samples considered positive, two were from black shouldered peafowl (Pavus cristatus), two were from American purple gallinules (Porhyrula martinica), and one each came from a Chestnut-bellied heron (Agamia agamia), an Emperor penguin (Aptenodytes patagonicus), a Rusted margined guan (Penelope supercilliaris), and a Roadside hawk (Rupornis magnirostris). Interestingly, all of the positive samples were obtained from adult birds. Phylogenetic analyses were based on a sequence of 146 nt after the removal of primer sites (Fig. 1). Most remarkably, nine mutations were revealed in the Pavus/2/BR/2009 (GU323507) sequence, despite many reports that consider the 3′ UTR region to be highly conserved among group 3 CoVs (Cavanagh et al. 2002). One hypothesis that could explain these mutations is the high density of different species of birds located in the same geographical area, which allows the virus to circulate among species (Jackwood et al. 2010). On the other hand, the natural habitat provides more distance between species, particularly between predators and prey. In addition, the sequences of the CoVs isolated from the two peafowl samples clustered separately from the British deposited sequences, as illustrated in Figure 1 (Cavanagh et al. 2002). Sequences within this cluster were relatively homogenous between two European samples (Fig. 1), in accordance with previous studies (Cavanagh et al. 2002).

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FIG. 1.

Minimum-evolution tree of coronaviruses (CoVs) based on a 146-nt fragment of the 3′ untranslated region from group 3 CoVs. Evolutionary distances were computed using the Tamura-Nei method and are expressed as the number of base substitutions per site. CoVs detected in captive wild birds by this study are denoted with an asterisk. Previously published CoV sequences from different sources were included for comparative purposes. GenBank accession numbers are shown in brackets. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Phylogenetic analyses were conducted in MEGA4. The scale bar indicates nucleotide substitutions per site.

Samples obtained from the Chestnut-bellied heron, two American purple gallinules, a Roadside hawk, and a rusted margined guan clustered together in a single clade, close to the sequence of the IBV D 207 strain (Figs. 1 and 2). In the present study of samples collected from 25 different species (kept in isolated locations within the zoological park), CoV RNA was detected in two Galliformes, two Ciconiiformes, one Gruiforme, and one Sphenisciforme. This demonstrates the high diversity in the susceptible species, as described previously (Woo et al. 2008). In fact, CoVs have been also detected in wildfowl species, rock doves, wild peafowl, waders, Amazon parrots, and some passerine species (Cavanagh 2005, Liu et al. 2005, Gough et al. 2006, Woo et al. 2008, Hughes et al. 2009). The hypothesis that wild free-flying birds present in zoos may often act as vectors flying between enclosures that house the exotic birds and transmitting infections is not fully understood.

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FIG. 2.

A comparison of part of the 3′ untranslated region from three CoVs isolated from chickens (prefixed IBV), one CoV from turkeys (prefixed TCoV), and two CoVs from pheasants (prefixed ph), with the same region identified in the eight CoVs isolated from the captive birds described in this study. Dots indicate that all the viruses had the same nucleotide at that position. Deletions are marked with an asterisk. Yellow shading indicates the mutations observed in the Pavus/2/BR/UNESP/09 sequence at a given position.

To understand the potential role of captive populations as reservoirs for CoVs, further studies are needed not only in South American zoo parks, but also worldwide. This is the first description of the molecular detection of group 3 CoVs related to captive birds in South America. The genetic diversity found in the black shouldered peafowl samples raises interesting questions about the 3′ UTR conserved region, its evolution, and viral circulation among different species inside zoo parks.