A genetic analysis of the complete spike glycoprotein of selected isolates of Massachustes and QX-like strains was carried out in order to predict molecular features by application of molecular and In silico technologies.
In the isolate CK/SWE/082066/10, amino acid phenylalanine substituted asparagine resulting in the loss of a potential N-glycosylation site at position 533. The five additional N-glycosylation sites found in QX strains may influence cellular tropism. The variation in N-glycosylation sites may affect survival and transnsmission of the virus. The small changes can lead to disturbance in folding and conformation of the molecule. The variations in N-glycosylation sites may affect the interaction with receptors and render a virus more susceptible to host innate immune system and lower recognition by antibody, hence affecting the virus replication and infectivity [39–42]. In a virus belonging to the Coronaviridae, lactate dehydrogenase elevating virus (LDEV), the loss or acquisition of N-glycosylation sites in the protein has been reported to determine changes of virulence and cellular tropism .
The presence of serine, threonine and tyrosine residues, with the right context for potential phosphorylation was investigated in the spike glycoprotein. The results have shown that the spike glycoprotein of IBV genotypes contained serine and thereonine peptides with potential for phosphorylation. The substitution of a serine by amino acid alanine at positions 563 caused loss of a potential phosphorylated peptide in strains CK/SWE/242/95 and CK/SWE/748/95. Wilbur et al. (1986) reported that N protein is the only structural protein that contained phosphorylation activity in coronaviruses . In this context we have observed potential phosphorylated peptides in the spike glycoprotein of IBV; however, this seems to be of no consequence for phosphorylation, since this property has not been identified in the spike which is well established as a glycoprotein.
The difference in cleavage site motifs of the spike glycoprotein of both genotypes, seem to indicate a correlation with geographical distribution of strains. It has been reported that serotypically and genotypically different IBV contain distinct cleavage site in the spike protein. However, the cleavage site sequence did not correlate with pathogenicity by comparing cleavage recognition sites in pathogenic and attenuated strains of IBV . The cleavage site motifs determined in Massachusetts and QX-like strains in present study are different from previously reported by Jackwood et al. (2001) . The replacement of arginine with histidine at amino acid position 537 in CK/SWE/748/95 and CK/SWE/748/95 may have influence antibody binding epitope. However, the functional meaning of this difference needs to be evaluated in proper biological assays. It has been reported that an IBV monoclonal antibody neutralization-resistant mutant (NR 18) had shown substitution of amino acid isoluecine for arginine in cleavage site motif which prevented cleavage and alter the conformation dependent monoclonal antibody binding epitope . In orthomyxoviruses and paramyxoviruses more basic residues around the cleavage recognition site of the envelope glycoproteins enhance the cleavability by ubiquitous host cell proteases, a characteristic which is associated with host cell range and increased virulence [44, 45]. However, the relevance of cleavage site to virulence was not observed in case of IBV spike glycoprotein .
The data regarding leucine-rich repeat regions of isolate CK/SWE/748/95 demonstrated that two LRR regions may result in strong folding capacity in the spike glycoprotein. It has been reported that leucine-rich repeat (LRR) is a structural motif of protein like a alpha/beta horseshoe fold and is responsible for protein-protein interactions [37, 38].
An additional palmitoylation site was found only in isolate CK/SWE/082066/10, indicating that it is strain specific. However, experimental studies are required to identify the effect of additional palmitoylation in the virus biology. In influenza virus, palmitoylation of the cytoplasmic domain of the hemagglutinin has been shown to increase its association with lipid mass . The deletion of palmitoylation sites in the hemagglutinin domain reduces hemagglutinin incorporation into the virion, thereby affecting the cellular trafficking . It has been reported that removal of palmitoylation sites in HIV-1 virus effected in the formation of virus with low levels of glycoproteins incorporation and reduced infectivity . Shen et al. (2009) reported attenuation of virus replication occurring due to mutations in the palmitoylation site in the non-structural protein nsp1 of Semliki Forest virus .
The predicted primary structures of the spike glycoprotein of both genotypic variants have shown a variable trend in physical properties such as isoelectric point, molecular weights and grand hydropathicity index. Grand average of hydropathicity index results suggested a hydrophobic nature of the spike glycoprotein in all variants. Cavanagh et al. (1986) reported that S2 sub-unit in N-terminal side of the membrane spanning hydrophobic domain of spike glycoprotein of IBV is susceptible to hydrolysis by many proteases .
The secondary structures prediction revealed that structures of the spike glycoprotein were found variable in both genotypic variants. It was also observed that there were no new structural domains and that no evolution has occurred in the structure of both genotypic variants. However, secondary structural variation in beta strands and residual coils have been observed between both genotypic strains.
A number of phylogenetic studies performed on the spike gene were mostly based on the partial sequences of S1 and N gene. Phylogenetic analyses based on the complete spike glycoprotein of avian coronaviruses demonstrated that IBV strains showed higher relatedness to turkey coronaviruses in comparison to other avian coronaviruses (Figure 1). This could be due to co-habitation of their host species. The appearance of a bat coronavirus branch close to these gammacoronaviruses suggests a possible common origin during earlier evolution of these viruses. The diversity of these viruses could have arisen from strong selective pressure resulting from mutations and recombinations that has been shown to occur in spike gene [14, 50, 51].
Phylogenetic analysis based on spike glycoprotein of avian coronaviruses and mammalian coronaviruses had revealed interesting trends. The mammalian beluga whale coronaviruses branched closer to the avian coronaviruses and two avian thrush and munia coronaviruses were related to the mammalian coronaviruses suggesting interspecies transmission. Furthermore, findings of the present study suggest that genetic diversity and molecular variation in sequences of spike glycoprotein of viruses made feasible the adaptation to new host species. Previously it has been reported that the RNA-dependant RNA polymerase gene and the whole genome of the beluga whale coronavirus was distantly related to IBV [52, 53]. Also a shift of tissue tropism in the virus has been reported [54, 55] resulting in infection of a wide range of avian host species especially those reared close to domestic fowl. For example, IBV cases were found in guinea fowl (Numida meleagris), Chinese peafowl (Pavo), teal (Anas) and partridge (Alectoris) .
The higher diversity in the avian, humans and bat-coronaviruses are due to continuous mutation, which may determine differences in tissue tropism. Previously it has been reported that diversity of coronaviruses due to a) infidelity of RNA-dependant RNA polymerase which lead to incorporation of nucleotide errors during replication [34, 57]; b) random template switching during RNA replication (copy choice mechanism) high rate of homologous RNA recombination events [4, 58]; c) extra plasticity nature in adjusting and modifying genes . These factors were responsible for the generation and diversification of variants and adaption to new hosts and ecological niches in the coronaviruses [53, 59].