Molecular Epidemiology and Evolution of Avian Infectious Bronchitis Virus

Mutation and recombination processes are involved in the genetic and phenotypic variations of RNA viruses, leading to the emergence of new variant strains, and give rise to virus population diversity to be modeled by the host, particularly by the immune system, as occurred with infectious bronchitis virus (IBV) in chickens. The consequence is a continuous emergence of new IBV variants with regard to pathotypes, serotypes, and protectotypes. Nucleotide sequencing and subsequent genetic analysis of the S1 and N protein gene sequences provide a fast and accurate method to classify and predict IBV genotype, and a powerful instrument to monitor phylogenetic and epidemiological evolution of IBV variants. Despite the use of vaccination programmes, infectious bronchitis has become a serious problem in Brazil. Thus, a significant number of IBV field variants have been identified circulating in the Brazilian commercial poultries between 2000 to 2006 and more recently in Argentina. These viruses seem to be indigenous, because they demonstrated a low genetic relatedness with the majority of the reference strains from North America, Europe and Asia, but were moderately to highly related one to another. In summary, indigenous field IBV variants were evolving and circulating in the field in Brazil and Argentina, and should be considered as initial candidates for protection against current IBV infectious in chickens. However, in vitro and in vivo studies are needed to determine the pathogenicity and immunogenecity of these new isolates, before defining a new vaccine strain.

multiple phenotypes in terms of pathotypes and immuno-types (Figure 1) (Cavanagh & Naqi 2003).Specifically, virus neutralization tests revealed the existence of a number of serotypes, which are poorly cross-protective (Cavanagh, 2007).
Coronaviruses have been classified into three groups, and IBV, together with turkey (TCoV), pheasant (PhCoV), goose (GCoV), duck (DCoV), and pigeon (PCoV) coronaviruses, and belong to Group 3 of Coronaviridae Family and order Nidovirales (Cavanagh & Naqi 2003, Cavanagh, 2005).The coronavirus groups were initially established on the basis of lack of antigenic relationship among the different groups, and nucleotide sequencing has largely confirmed these groupings (Masters, 2006).
Central to population genetics is understanding how the five main forces of evolutionary change -mutation, recombination, natural selection, genetic drift, and migration -interact to shape the genetic structure of populations.These same forces are also essential to understand RNA viruses evolution (Moya et al., 2004).
IBV, like many other RNA viruses, as well as the coronaviruses, has a high error rate during the transcription of its genomes (Lai & Cavanagh, 1997), producing a quasispecies phenomenon, where many different viral genotypes co-circulate in the host, with each virus potentially having different levels of fitness for the host environment (Domingo et al., 1985).In fact, the quasispecies phenomenon has been reported to occur among coronaviruses, and it is considered important for the evolution and persistence of these viruses, because it may favor the emergence of new variants in nature, because it may provide mechanisms for virus-escaping from host immune responses (Rowe et al., 1998, Kiss et al., 2000, Nix et al., 2000) (Figure 2).Thus, IBV is continuously evolving, producing new antigenic and/or pathotype variants (Lee, 2002) (Figures 1 and 3).In order to illustrate the importance and frequency of these phenomena since the first isolation of IBV in the 1930s, over 50 serotypes or variants have been described worldwide since then (Cavanagh & Naqi, 2003;Lee & Jackwood, 2000).

GENOMIC STRUCTURE, VIRAL PROTEINS, AND BIOLOGICAL ACTIVITIES OF IBV
The genome of IBV consists of a 27.6 kb single stranded positivesense RNA molecule including 7 to 10 open reading frames (ORFs) that encode both structural and non-  ).The remainder of the genome encodes four structural proteins: the spike (S) glycoprotein, the small envelope (E) protein, the membrane (M) glycoprotein, and the nucleocapsid (N) protein, as well as five small non-structural accessory proteins: 3a, 3b, 3c, 5a, and 5b, which are not required for in-vitro replication, but may play a role in pathogenesis (Casais et al., 2005, Hogson et al., 2006).The genome has the following general organization: 5'UTR -polymerase gene -S-3a,b,c -(E)-M-5a,b-N -UTR 3' (Lai & Cavanagh, 1997, Cavanagh, 2007) (Figure 2).Gene S consists of one ORF that encodes the spike glycoprotein (S), which is posttranslationally cleaved into the subunits amino-terminal S1 (92-kDa) and carboxyl-terminal S2 (84-kDa), comprising approximately 500 and 600 amino acids, respectively (Cavanagh, 1983;Cavanagh, 2007).In the mature virion, S2 associates with S1, anchoring the S1 protein to the membrane to form the multimeric coiled-coil S protein (Cavanagh, 2007).Gene N has one ORF, encoding the nucleoprotein, which, together with the genomic RNA, forms the helical nucleocapsid (Collison et al., 1992;Sapats et al., 1996).This protein is highly conserved, differing among distinct IBV isolates by only 2 to 6% at the amino acid level (Williams et al., 1992).
IBV's S and N proteins have relevant biological and immunological functions (Figure 2).The S glycoprotein can be divided into three structural domains: a large external domain, which is further divided into two subdomains S1 and S2, a transmembrane domain, and a short carboxyl-terminal domain (Cavanangh, 1983, Lai & Holmes, 2001).The S1 sequence is more variable, and mutations in this sequence have been associated with altered tissue tropism and antigenicity in IBV strains (Cavanagh & Naqi, 2003).In fact, the S1 sequences from different strains vary significantly, usually by between 2 and 25% at the amino level, whereas the S2 subunit is more conserved (Cavanagh et al., 1992(Cavanagh et al., , 1997;;Kant et al., 1992;Koch et al., 1990).As to its biological properties, the S1 glycoprotein is the main inducer of virus-neutralizing antibodies, and regulates several other important activities, such as attachment to cells, fusion of the viral envelope with host-cell membranes and, sometimes, cell to cell fusion (Kant et al., 1992, Koch et al., 1990, Lai & Holmes, 2001) (Figure 2).IBV nucleocapsid protein is a phosphoprotein containing 409 amino acids, and it is well-conserved across various IBV strains (Williams et al., 1992) and important for cell-mediated immunity (Seo & Collison, 1997, Seo et al., 1997).It forms a protective shell that packages the viral genomic RNA, and it is also thought to participate in viral RNA replication and transcription.Specific packaging of viral genetic material is usually performed via recognition of a particular nucleotide sequence by a nucleocapsid protein (Fan et al., 2005) (Figure 2).
Therefore, variations in S1 and N genes, in particular, are believed to be of critical importance for emergence of variants due to their role in virus replication and immunity, and hence S1 and N genes and derived proteins are more frequently used to determine the relatedness of emerging IBV.

EVOLUTIONARY CHANGES OF INFECTIOUS BRONCHITIS VIRUS AND THEIR IMPLICATIONS FOR EPIDEMIOLOGY AND CONTROL OF THE DISEASE
As previously mentioned, IBV infection is worldwide distributed and it is extremely difficult to control because multiple serotypes and variants of the virus are continuously emerging and are not cross protective (Cavanagh & Naqi, 2003, Cavanagh, 2007).Moreover, coronaviruses, including IBV, have been shown to be a mixture of genetic mutants within an isolate, and
Additionally, molecular studies have shown that a new serotype or variant can emerge as a result of only a few changes in the amino acid composition in the S1 part of the virus spike protein, while most of the virus genome remains unchanged (Cavanagh, 2007).This could be due to immunological pressure caused by the widespread use of vaccines, to recombination as a consequence of mixed infections, or to a reduction of dominant serotypes as a result of vaccination, allowing other field strains to emerge (Lee, 2002;Liu et al., 2006) (Figure 4).
The epidemiological investigation of IBV infections has demonstrated that the spreading of a strain from one area or country to another could be due, at least in part, to its improper introduction by the trading of birds, by migratory wild birds, or by the use of attenuated vaccines (Cavanagh, 2005;Liu et al., 2006) (Figure 5A).A very interesting aspect of IBV epidemiology is the presence and dissemination of various IBV serotypes in different continents.A relevant number of emergent serotypes in North America did not spread to other continents, and similarly, European, Australian, and Asian serotypes apparently did not spread elsewhere (Liu et al., 2006).However, there are indications that IBVs detected in others countries, such as in China, derive from genetic changes both in IBV populations that existed before vaccination started and in the viruses introduced through live vaccines using attenuated strains from China and other continents, leading to the emergence and co-circulation of strains with various genetic and phenotypic differences (Liu et al., 2006).
A recent review on coronavirus diversity and genomics (Woo et al., 2009) speculated that the ancestor of the present coronaviruses infected a bat, and it jumped from the bat to a bird, or alternatively, it infected a bird and it jumped from the bird to a bat, evolving dichotomously.Or else, the bat coronavirus may have jumped to another bat species, giving rise to group 1 and group 2 coronaviruses, evolving dichotomously.These bat coronaviruses then jumped to other bat species and other mammals, including humans, with each interspecies-jumping evolving dichotomously.On the other hand, bird coronaviruses jumped to other species of birds, and occasionally to some specific mammalian species, with each interspecies-jumping evolving dichotomously, giving rise to group 3 coronaviruses (Woo et al., 2009) (Figure 5B).
IBV evolution is a complex and poorly understood process (Ignjatovic et al., 2006;Vijaykrishna et al., 2007).However, investigations carried out to date highlight the role of three factors: (1) lack of RNA polymerase proofreading, leading to replication errors in RNA genomes with mutation at the order of 10 -4 ; (2) interference of continuous use of live and often multiple attenuated vaccines formulated with different IBV strains, and (3) immune pressure exerted on circulating viruses by the constant presence of partially immune bird populations.The acquisition of the replicase machinery may have improved the low fidelity of RNA replication to allow genome expansion and give rise to the ancestors of small and, subsequently, large nidoviruses, including the coronaviruses (Gorbalenya et al., 2006).
Genomic alterations affecting coronaviruses probably act in concert and involve point mutations, deletions, insertions, and recombination to generate new virus variants, particularly with regard to S1 and N genes (Cavanagh et al, 1992;Ignjatovic, et al., 2006;Jia et al., 1995;Kusters et al., 1990;Lee & Jackwood, 2000;Wang et al., 1993).Interestingly, evolutionary and mutation rates of IBV isolated from vaccinated birds reached 2.5 and 1.5% per year, respectively, in the hypervariable regions of the S1 glycoprotein, but, in
Therefore, the continuous emergence of new IBV serotypes has hampered the development of appropriate control programs due to antigenic variation and the low degree of cross-protection observed among IBV serotypes (Dolz et al., 2008).This emphasizes the need for prompt and precise diagnosis, mainly to identify new variants of this viral agent and to compare them with the existing vaccine strains (De Wit, 2000).Nucleotide sequencing, followed by phylogenetic analysis of S1 protein gene sequences, offers a rapid and accurate method to identify the IBV genotype, as well as to predict IBV serotype, thereby contributing to evaluate phylogenetic and epidemiological evolution of IBV strains (De Wit, 2000; Lee and Jackwood, 2001;Moore et al., 1997;Wang & Huang, 2000).It was demonstrated that mutation and recombination processes are involved in IBV genetic variation and subsequent evolution by a mechanism that leads to the emergence of new variant strains and the generation of virus population diversity (Jia et al., 1995;Kottier et al., 1995;Lee & Jackwood, 2000;Wang et al., 1997) (Figure 3).However, very distinct epidemiological behaviors among these new strains have been described (Figure 5).It is intriguing that some emergent viruses rapidly spread to other geographic areas and become prevalent, whereas others remain restricted to the region of origin.Therefore, monitoring the absence of immunity induced by vaccines, IB viruses evolve at a much slower rate (0.3%) (Lee & Jackwood, 2001;McKinley et al., 2008) (Table 1).

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IBV subpopulation dynamics in a specific region over time may reveal molecular features that contribute to the different fitting and spreading abilities of IBV strains (Dolz et al., 2008).Moreover, the effect of recombination on the generation of IBV antigenic variants capable of escaping from the immune responses of the vaccinated chickens can be also mediated by S1 and N genes (Figure 6).Considering that the 5' region of S1 protein contains most of the neutralizing epitopes (Kant et al., 1992;Koch et al., 1990;Lai & Holmes, 2001), and that the 3' region of N protein contains protective CTL epitopes (Seo & Collison, 1997;Seo et al., 1997) as well as the most immunogenic linear B-cell epitopes (Seah et al., 2000), inter-strain recombination between these genes could act as a relevant mechanism for the generation of genetic and immuno-phenotypic variants that result in the selection of better adapted IBV strains, exhibiting distinct antigen-types, protectotypes, and pathotypes (different tissue tropisms, and increased or reduced virulence).Thus, a successful IBV escape mutant must have altered critical epitopes in these viral proteins, in order to persist, and/ or cause disease in immunized host chickens (Yu et al., 2001).
In summary, in the context of viral evolution, recombination is a fast and efficient way to get rid of accumulated deleterious changes in the genome and to create or spread beneficial combinations of mutations in an efficient manner (Lee, 2002) (Figure 6).

MOLECULAR EPIDEMIOLOGY AND EVOLUTION OF IBV IN BRAZIL
Brazil is currently one of the major poultry-producing countries in the world and in the last few years infectious bronchitis has become a serious problem and is considered endemic.This disease is controlled in Brazil mainly by vaccination using live attenuated or inactivated vaccines mostly based on Massachusetts strain.Neverthless, outbreaks of IB are still occurring in vaccinated flocks, indicating that, most probably, variant isolates of a different serotype to the vaccine strain were emerging.
In 1957 the first isolation of IBV was recorded in Brazil (Hipólito, 1957), and despite the official introduction of IBV vaccination in 1979 with strains H52 and/or H120, outbreaks have continued (Ito, 2006).Apparently, most Brazilian IBV field isolates recovered until 1989 were classified by cross-neutralization test as Mass serotype (Ito, 2006).Di Fabio et al. (2000) investigated by cross-neutralization tests of tracheal organ cultures a group of 15 IBV field isolates collected in Brazilian commercial flocks, which had experienced a variety of IB-like conditions, including respiratory disease, digestive and kidney problems, and/or drop in egg production.One of the isolates was associated to the Massachusetts (Mass) serotype and the other fourteen were classified in at least four antigenic groups, all distinct from those previously described in other countries.
Twelve Brazilian IBV field isolates were recovered from different IB outbreaks affecting commercial broiler or layer flocks located in South andSoutheast Brazil between 1988 and2000.These viruses have amplified the S1 gene by RT-PCR and were analyzed by restriction length polymorphism (RFLP) of the entire S1 gene.Additionally, nucleotide sequences of the 5'part of the S1 gene containing 399 bp, including hypervariable regions 1 and 2, were also obtained and analyzed in comparison with reference IBV strains, including H120 vaccine strains.The S1 phylogenetic analysis, using either the partial nucleotide or amino acid deduced sequences of these twelve isolates, supported the RFLP findings, based on the use of the enzymes HaeIII, XcmI and BstyI on the entire amplified product of S1 gene and separated these viruses into five genotypes, identified by five clades in the dendrogram.The identity values for the S1 sequence of the isolates classified by RFLP as Mass genotype along with the H120 strain, were high and ranged from 96.4 to 100%, characterizing an individualized cluster of these viruses (clade I).There is one isolate with a unique XcmI-RFLP profile, which displayed lower relationship with Mass genotype and represented a distinct genotype and was classified in a separate branch (clade II).The other six field isolates, identified by HaeIII-RFLP as three different genotypes, were even less related to the Mass genotype, exhibiting lower identity values to Massachusetts-related strains.However, close relationships were detected for the viruses belonging to each one of the three groups, resulting in the separation by phylogenetic analysis into clade III (Genotype A by RFLP), clade IV (Genotype B by RFLP) and clade V (Genotype C by RFLP) (Montassier et al., 2006(Montassier et al., , 2008) (Figure 7).
Among the Brazilian IBV variants, and classified more distant to the Massachusetts Genotype, there are two viruses isolated in 1988 and two in 2000 from birds with IB respiratory signs.In an additional phylogenetic analysis, including 5'-S1 gene sequences of reference IBV strains and another set of IB viruses isolated more recent in Brazil (2004 and2006), these viruses displayed low genetic relationship with most of the reference strains of IBV from North America, Europe and Asia, but exhibited an intermediate to high relationship with the new set of Brazilian isolates recovered between 2004 and 2005 from poultry flocks with enteric and reproductive problems.It is also noteworthy that the viruses isolated earlier, in 1988, were placed, after the phylogenetic analysis, in a branch more distant from those branches clustering the variant viruses, isolated more recently, in the years of 2000, 2004and 2005 (Figure (Figure 7A).Additionally, two of these new Brazilian variants were clustered together with three Argentinean isolates recovered from broilers and layers during different outbreaks in commercial poultry flocks in different geographic regions of Argentina from 2001 to 2008.All these Argentinean and Brazilian IBV isolates shared 93.4% of similarity in their amino acid sequences (Rimondi et al., 2009) (Figure 7B).Therefore, these variant viruses seem to be indigenous of Brazil and Argentina, and may share a common ancestor.They are evolving in the field by infecting either vaccinated, or non-vaccinated chickens from commercial poultries in these countries (Figure 8).
To date, the obtained data have also demonstrated that a Brazilian variant genotype has been circulating and evolving in the field since at least as early as 1988, but there are still few scientific studies on the genetic, antigenic, and pathotype variation of serotypes and dynamics along time of IBV genotypes/phenotypes of  (Rimondi et al., 2009).These viruses were moderate to high genetic related one each other (86.4 to 99.2% of identity) and the groups were supported by high bootstrap values (Montassier et al., 2006, Rimondi et al., 2009).

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a specific region, and particularly in Brazil.Although these viruses may be the reason of IB frequent outbreaks in that country, it is also essential to determine the immunotypes (serotypes and protectotypes) and the pathotypes (tissue tropism and virulence properties) of these variants in order to fully characterize them.Therefore, the biological and epidemiological behaviors of these variants need to be further studied, contributing to define a more effective programs for IB control, considering that the inefficacy of the current control methods based only on monovalent vaccination with the Massachusetts serotype vaccine is the main issue to be solved.

Figure 1 -
Figure 1 -Genetic and phenotypic variations are common features in the evolution process of infectious bronchitis virus strains and lead to important antigenic and pathotype variability.

Figure 3 -
Figure 3 -Role of mutation, recombination and selection in the generation and evolution of quasispecies of IBV strains.

Figure 4 -
Figure 4 -Schematic diagram of the possible evolutionary pathway and emergence of a new antigenic variant (/serotype) from a parental strain of IBV (Adapted from Lee, 2002).

Figure 5 -
Figure 5 -Proposed epidemiology and transmission of IBV among wild birds and poultry (A).Postulated ecology of Coronaviruses.Hypothetical interspecies transmission and evolutionary pathways.Solid and dashed lines represent confirmed and hypothetical interspecies transmission (B) (Adapted from Woo et al., 2009).

Figure 7 -
Figure 7 -Dendrograms highlighting a group of indigenous IB viruses isolated from Brazil (IBVPR01, IBVPR02, IBVPR05 and IBVPR06) (Montassier et al., 2006) (A) and Argentine (B) composed by Cluster C derived from phylogenetic analysis of 3 Argentinean and 2 Brazilian IB indigenous variants based on the 5'-part of S1 glycoprotein gene (nucleotides 685 to 836)(Rimondi et al., 2009).These viruses were moderate to high genetic related one each other (86.4 to 99.2% of identity) and the groups were supported by high bootstrap values(Montassier et al., 2006, Rimondi et al., 2009).

Figure 8 -
Figure 8 -Hypothetical interactions and evolutionary pathways for new IBV variants from Argentina and Brazil.The possible consequences on immunoprophylaxis scenarios of IBV infection in these countries are presented.
Montassier HJ Molecular Epidemiology and Evolution of Avian Molecular Epidemiology and Evolution of Avian Molecular Epidemiology and Evolution of Avian Molecular Epidemiology and Evolution of Avian Molecular Epidemiology and Evolution of Avian

Table 1 -
Lee, 2002) of the evolution of two strains of IBV submitted to different immune selective pressures (Adapted fromLee, 2002).Due to the high variability and important biological properties of the S1 glycoprotein, antigenic evolution in IBV has been primarily associated with changes in the sequence of the S1 glycoprotein, which contains Molecular Epidemiology and Evolution of Avian Common ancestor of IBV????