To detect the presence of infectious bronchitis virus or avian coronavirus, a nested reverse transcriptase PCR (RT-PCR) method was developed with the aim of amplifying a fragment of 530 bases, comprising the gene coding S1 protein. In the first step, all samples were submitted to RNA extraction, RT-PCR, and nested PCR. Next, only the positive nested-PCR samples were propagated in specific-pathogen-free (SPF) embryonated chicken eggs for virus isolation. Positive samples were then sequenced and analyzed using a molecular phylogeny approach. Tracheal swab samples were collected from 23 different domestic chickens distributed in three regions of Brazil, in the period between 2003 and 2009. Also analyzed were six swab samples (tracheal and cloacal) from asymptomatic pigeons (Columba livia), caught in an urbanized region in southeastern Brazil. The study revealed two major phylogenetic groups: one clustered with the Massachusetts vaccine serotype and another joined with the D207 strain. Interestingly, samples grouped with the Connecticut and Arkansas serotypes were also found. Pigeon isolates clustered with the Massachusetts serotype showed significant similarity (close to 100%) to those obtained from chickens. Only one pigeon isolate was seen to be grouped with the Connecticut serotype, and no correlation was observed between sample grouping and region origin. Understanding the diversity of genotypes and eco-epizootiology of the disease in different environments is expected to be helpful for vaccine production aimed at the main circulating variants. In this respect, one could also expect benefits in the management of other bird species that may act as avian coronavirus reservoirs.
Avian infectious bronchitis virus (IBV), a member of the family Coronaviridae, order Nidovirales, is a highly infectious pathogen of domestic fowl 6,7,43. IBV is an enveloped virus that replicates in the cell cytoplasm, and its genome is constituted by a nonsegmented positive-stranded RNA of 27.6 kb 3. All coronaviruses maintain a set of essential genes, including those that encode the polymerase (Pol), spike (S), small membrane (E), membrane (M), and nucleocapsid (N) proteins in the invariable order 5′-Pol-S-E-M-N-3′ and a 3′ untranslated region (UTR) 6,7. In addition to these essential genes, IBV contains group-specific or accessory genes that encode small proteins 23. Although the coronaviruses are traditionally separated into three groups 4,23 based on genetic and antigenic characteristics, many other groups and subgroups have recently been proposed 13,18,40,46. These viruses have been associated with diseases in several warm-blooded animals, including humans 22,42. Coronaviruses from group I and II have been found to infect several mammalian species, including humans, pigs, cows, dogs, horses, cats, and rodents. Group III viruses have been found to infect poultry. This group includes the chicken IBV, the turkey coronavirus, and the pheasant coronavirus 22,43. Although IBV indeed causes respiratory disease, it also replicates at many nonrespiratory epithelial surfaces, where it may cause pathology 6, and represents one of the most economically significant diseases of the intensive poultry industry. In young chicks, respiratory disease or nephritis lead to mortality, reduced weight gain, and condemnation at processing; whereas, in chickens of laying age, the disease is subclinical and results in reduced egg production and aberrant eggs 6,32.
Since IBV was first described by Schalk and Hawn in the 1930s 14, many serotypes have been identified worldwide and new variants have arisen, many of them as a result of vaccination programs 28. Vaccines are generally effective in controlling the clinical disease; however, escape mutants or variants continue to cause clinical disease and production problems in vaccinated flocks 8,20. The continuing appearance of new IBV variants is associated with the high evolution rate, expressed as the accelerated rate at which viable mutations accumulate in the genome 12,24,25.
Coronavirus has also been isolated from wild bird species and racing pigeons, which could constitute an important environment for virus evolution 17,22,31,46. The other problem is the possibility of recombination events within coronaviruses, as may be the case for the severe acute respiratory syndrome (SARS) virus. In fact, some authors have argued that there is evidence for recombination events in the evolution of this virus, involving both mammalian and avian coronaviruses 36,37.
Molecular epizootiology can be very important to understand the coronavirus dynamics in various correlated avian species and to improve its control. Avian populations, such as, e.g., pigeons, which are asymptomatic for respiratory diseases, could be critical in this context, because they can transport the virions. In this way, they provide an important environment for virus evolution. The main objective of the present study was to better understand the genetic diversity aspects of avian coronavirus in Brazilian commercial poultry and pigeons. The study was based on molecular characterization and phylogenetic analysis performed on partial sequences of the S1 gene of IBV isolated from chicken (Gallus gallus domesticus; historical 5-yr series) and coronavirus from feral pigeon (Columba livia).
MATERIALS AND METHODS
In the period from 2003 to 2009, a total of 23 positive samples, isolated from 102 tracheal swab pools from IBV symptomatic chicken, were obtained from the main poultry-producing regions of Brazil; namely, the southern 11, southeastern 6, and northeastern 1 regions (Table 1). In addition, there were six positive samples from 12 pigeons with no symptoms of IBV disease (cloacal and tracheal swabs) captured at the city of Campinas (São Paulo State, southeastern region of Brazil) in 2006 and 2007. These pigeons were identified and released after completion of tracheal and cloacal swabs. All swabs were resuspended on 0.2 ml of minimum essential medium (MEM), from which 0.1 ml was used for further molecular studies and subsequent inoculation in embryonated specific-pathogen-free (SPF) eggs (positive samples by reverse transcriptase (RT)-PCR).
Brazilian samples utilized in this study with their respective regions of origin, host, and GenBank number.
RNA extraction and viral nucleic acid amplification
The swabs were resuspended in 0.1 ml of MEM in an Eppendorf tube and centrifuged at 800 × g for 5 min. RNA was extracted from the supernatant using a High Pure Viral Nucleic extraction kit™ (Roche Diagnostics™, Mannheim, Germany) and the cDNA was synthesized using a High Capacity cDNA kit (Applied Biosystems, Foster City, CA). Both procedures were performed according to the manufacturer's instructions for use with random hexamers. All samples were investigated for IBV through the amplification of specific genome fragments. The amplification of the variable region of the S1 protein gene was the target for the polymerase chain reaction.
The first step of nested RT-PCR was carried out using forward primer S7 (5′-TACTACTACCAGAGTGC(C/T)TT-3′) annealing at the 20,455 IBV genome position (Massachusetts 41 type) and a reverse primer, S6 (5′-ACATC(T/A)TGTGCGGTGCCATT-3′) annealing at the 21,008 position 2 with amplified products of 572 bp. PCR was carried out in a 50 μl mixture containing 5 μl of 10× PCR buffer (10 mM Tris-HCl pH 8.0, 50 mM KCl), 1.5 μl of 0.2 mM MgCl2, 1.5 μl of a 10 mM dNTP mixture, 2.5 μl of each primer (10 mM), 5 μl of cDNA, and 0.2 μl (5 U/μl) of Platinum™ DNA Polymerase (Invitrogen Ltd., Carlsbad, CA). The amplification was preheated for 5 min at 95 C, followed by 35 cycles, each consisting of 1 min at 95 C (denaturation), 1 min at 55 C (annealing), and 1 min at 72 C (extension). After completion of the 35 cycles, a final extension of 7 min at 72 C was performed.
A second step of nested PCR was carried out with the amplified products of the first-round using forward primer S9 (5′-ATGGTTGGCATTT(A/G)CA(C/T)GG-3′; 20,486 genome position) 2 and the reverse primers S5 (5′-GTGCCATTGACAAAATAAGC-3′; 20,995 genome position) 2. The nested PCR was performed using nested primers S9-S5 corresponding to the amplified products of 530 bp. The amplifications were carried out in a thermal cycler PCR System 9,700 (Gene Amp, Applied Biosystems, Perkin-Elmer, Carlsbad, CA). The PCR products were run on 1% agarose gel and visualized under UV light after staining with ethidium bromide.
Passage in embryonated SPF chicken eggs
The positive samples, after RT-PCR and sequencing, were inoculated 0.1 ml onto the chorioallantoic membrane (CAM) of embryonated SPF chicken eggs (9 days old). The eggs were incubated and observed daily for viability. After 1 wk the embryos were evaluated for lesions typical of IBV (stunting and curling). This process was repeated five times. In all passages, control samples were taken for molecular investigations (RT-PCR and sequencing).
Sequencing and phylogenetic analysis
PCR products were sequenced three times each, in both the forward and reverse directions using an ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems). The BioEdit software, version 220.127.116.11 16, was used to manipulate the retrieved nucleotide and amino acid sequences. The sequence alignments were performed using the Clustal W software version 1.83 41 using full alignment and 1000 total replications on the bootstrap in order to ensure a higher level of confidence for the analysis 41. Phylogenetic analyses were performed using neighbor-joining as implemented in the MEGA version 4 software package 39 based on the Kimura two-parameter distance estimation method. Bootstrap resampling was performed for each analysis (1000 replications). Reference IBV nucleotide sequences were retrieved from the GenBank database (Table 2), including the complete Massachusetts type genome (accession number AY851295) utilized to estimate the nucleotide positions. The IBV nucleotide sequences obtained have been submitted to GenBank.
Names, hosts, and accession numbers of the different coronavirus strains from the GenBank used in this study.
Clusters and similarity of studied S1 fragment from different samples
The chicken samples demonstrated two principal groups, one similar to the Massachusetts strain (IBV/Brazil; 2006/UNICAMP788, 2008/UNICAMP836, 2008/UNICAMP861, 2008/UNICAMP818, 2009/UNICAMP901, 2009/UNICAMP897, 2008/UNICAMP890, 2008/UNICAMP832, 2008/UNICAMP820, 2008/UNICAMP857, and 2008/UNICAMP821) and the other different from vaccine strains used in Brazil (IBV/Brazil; 2007/UNICAMP801, 2008/UNICAMP846, 2003/UNICAMP31422, 2004/UNICAMP31298, 2004/UNICAMP706, 2004/UNICAMP703, 2009/UNICAMP940T, 2008/UNICAMP882, 2007/UNICAMP810, and 2008/UNICAMP816), which grouped to the D207 strain. Five pigeon samples were similar to the Massachusetts group in the chickens (Columba/Brazil; 2006/UNICAMP63C, 2006/UNICAMP64C, 2007/UNICAMPT6, 2007/UNICAMP67T, and 2007/UNICAMP65T). One pigeon sample (Columba/Brazil/2007/UNICAMPT66) and another from chickens (IBV/Brazil/2007/UNICAMP 805) were similar to the Connecticut strain (not used in Brazil as vaccine), and one sample from the chickens (IBV/Brazil/2008/UNICAMP 830) was very different from the others and grouped with the Arkansas strain (Fig. 1).
The similarity of the S1 fragment nucleotide sequence from coronavirus isolated from pigeon and chicken ranged from 100% to 51.7% (100% to 22.6% for the amino acid sequence). In just the chickens, this range was from 100% to 71.8% (100% to 50.6% for the amino acid sequence), while in pigeons it was from 100% to 60.8% (100% to 33.3% for the amino acid sequence). When the intercluster similarity was considered, the Massachusetts type ranged from 77.3% to 69.1% (62.2% to 46.2% for the amino acid sequence) for the D207 group, 94.1% to 60.4% (88.6% to 33.9% for the amino acid sequence) for the Connecticut type, and 72% to 67.4% (57.4% to 49% for the amino acid sequence) for the Arkansas type.
With regard to the isolation chronology, the Massachusetts type was isolated from 2006 to 2009 in chickens and from 2006 to 2007 in pigeons. The D207 type was isolated since 2003 only in chickens. No correlation was observed between sample grouping and region origin.
Passage in SPF eggs
All samples used in this study, both those of chicken and pigeon, when passed in embryonated SPF eggs, caused lesions consistent with IBV, notably dwarfing and body curling.
Samples and country region
When the isolates were considered by region of origin, it was observed that of those from the southern region (n = 9), six grouped with the Massachusetts type and three with the D207 cluster. Of those from the southeastern region (n = 11), four grouped with the Massachusetts type, five with the D207 cluster, one with the Arkansas type, and one with the Connecticut type. Among the three samples from the northeastern region, two grouped with the Massachusetts type and one with the D207 cluster.
The genetic diversity found in the viruses isolated from chickens in this study was relatively higher than that reported by other molecular studies concerning Brazilian samples in which only viruses corresponding to the D207 group were described 44,45. These studies showed no Massachusetts group, perhaps due to the screening method chosen to evaluate only the samples that were amplified by RT-PCR of the UTR of their genomes. Thus, it is interesting to consider the possibility of differences in these and others genome regions among the different virus clusters found in Brazil; certainly others studies should be performed to verify this hypothesis. In a characterization of IBV isolated after an outbreak in Brazil in the late 20th century, a variety of serotypes was also observed including the presence of the Massachusetts type 10. Another retrospective Brazilian IBV study, using the restriction fragment length polymorphism technique and predicted N protein amino acid composition, also showed great diversity (classified in six genotypes), mainly after official vaccination 1. Besides these Brazilian cases, virus diversity has also been described in other countries 5,19,21,27,30,33. Recent studies involving the complete sequencing of coronavirus isolated from chickens have demonstrated the existence of viruses with different genomic organizations 11,26,29.
Of the 23 virus strains from chickens used in this study, 11 grouped with the Massachusetts serotype. This may represent a possible vaccine origin virus used for production of attenuated vaccines currently in Brazil. Our findings have shown lesions characteristic of IBV when passaged in embryonated SPF eggs (stunting and curling) and isolated from chickens with swollen head syndrome. All six isolates from pigeons have also caused the same injury when passed in chicken eggs SPF. Other investigations in vivo for studying the pathogenicity of these isolates for chickens certainly need to be made. In any case, even if the viruses isolated were from a vaccine origin, it is especially important to consider that most coronaviruses isolated from healthy pigeons showed regions of the gene coding for S1 with a similarity close to 100% with IBV isolates. One can conjecture that pigeons or other synanthropic birds, as well as wild ones, could actively participate in the eco-epidemiology of IBV.
These avian species could constitute a host range in which the virus can evolve, as well as a space for recombination between the wild-type coronavirus and IBV vaccine. As a consequence, new variants with different pathogenic potential may be produced 15. For example, in one study, coronavirus isolates with high similarity to the Massachusetts serotype were nonpathogenic to peafowls but pathogenic for chickens 38. In an earlier study, coronaviruses were encountered in seven wild birds (four ducks, one swan, one red knot, and one Eurasian oystercatcher); of these, three of the duck samples and the one from the swan grouped with the H120 (Massachusetts) vaccine strain, and those authors also conjectured about the possibility of these being revertant attenuated vaccine strains 17. Another study involving the S1 coronavirus gene isolated from pigeons also showed genetic and antigenic similarities with the Massachusetts genotype, but in this case the virus causes pancreatitis in this species 35. Yet another study found coronavirus different from the Massachusetts type in pigeons as well 22.
A small number of isolates have appeared only once since 2004, namely IBV/Brazil/2007/UNICAMP805, which grouped with the Connecticut type, and a sample IBV/Brazil/2008/UNICAMP830, which grouped together with the Arkansas type. This is noteworthy, because, to our knowledge, it has never been reported before. In Brazil, Massachusetts-type virus is used in preventive live vaccine. One explanation for the occurrence of the Arkansas and Connecticut serotypes could be the use of unauthorized live vaccines. Another important finding is the absence of isolated virions in pigeon samples within the D207 group. This could mean either that the virions are not adapted to pigeon or the number of samples was not large enough.
In this study the samples were collected in the main regions producing chicken and eggs in Brazil (south, southeast, and northeast) and in all these the presence of isolates from groups D207 and Massachusetts was observed. This shows that there is apparently no predominance of one or another group among the different regions of the country. Only in the southeastern region were isolates observed that were grouped with Arkansas and Connecticut types, an occurrence that is difficult to assess since only two viruses were found.
An overall investigation of the eco-epizootiology of IBV must include the possibility of virus evolution or transport by other animal species, especially those that often coexist in the farm environment. Moreover, the dynamics of the attenuated vaccine virus in the environment must also be considered for the control of avian infections. A better understanding of these issues would aid in the development of more effective vaccines and vaccination programs, as well as improved livestock health management practices. Also, in light of recent examples such as SARS and the newly discovered bat and wild mammal coronaviruses 34,40,46, a direct benefit would be the decreased emergence of new coronaviruses that pose a threat to the health of poultry and humans.