Turkey coronavirus is more closely related to avian infectious bronchitis virus than to mammalian coronaviruses: A review

Turkey coronavirus (TCoV) is the cause of an acute highly contagious enteric disease of turkeys. In recent years, TCoV has been increasingly recognized in North America as an important pathogen of young turkeys, resulting in economic loss due to impaired growth and poor feed conversion. While the epidemiology and pathogenesis of TCoV have been extensively studied, TCoV remains one of the least characterized of the known coronaviruses. Avian and mammalian coronaviruses have been subdivided into distinct antigenic/genotypic groups; however, classification of TCoV has been controversial. Previous studies indicated that TCoV was closely related to bovine coronavirus and other group 2 mammalian coronaviruses, but more recent antigenic and genome sequence analyses contradict these findings and, instead, provide evidence that TCoV is closely related to avian infectious bronchitis virus (IBV). Additionally, experimental studies have indicated that the host range of TCoV, once thought to be restricted to turkeys, includes chickens. These studies have raised additional questions regarding the classification of TCoV; particularly, whether IBV and TCoV are taxonomically distinct viruses, or whether TCoV is merely a variant of IBV. Sequence analyses of TCoV have given credence to the idea that TCoV is a variant of IBV, as these studies have shown that TCoV and IBV are very closely related. However, these studies have been limited to only three TCoV strains and relatively small portions of the TCoV genome. TCoV is readily distinguished from IBV based on antigenic and biological differences, and these differences suggest that TCoV should be considered a distinct virus species. Additional studies will be needed to better define the relationship between TCoV and IBV, and to resolve this taxonomic question. Based on our current understanding, it seems prudent to consider TCoV and IBV as distinct virus species that share a close phylogenetic relationship and together comprise group 3 of the coronavirus major antigenic groups.     

Recombinational histories of avian infectious bronchitis virus and turkey coronavirus

Phylogenetic analysis of complete genomes of the avian coronaviruses avian infectious bronchitis (AIBV) and turkey coronavirus (TCoV) supported the hypothesis that numerous recombination events have occurred between these viruses. Although the two groups of viruses differed markedly in the sequence of the spike protein, the gene (S) encoding this protein showed no evidence of positive selection or of an elevated mutation rate. Rather, the data suggested that recombination events have homogenized the portions of the genome other than the S gene between the two groups of viruses, while continuing to maintain the two distinct, anciently diverged versions of the S gene. The latter hypothesis was supported by a phylogeny of S proteins from representative coronaviruses, in which S proteins of AIBV and TCoV fell in the same clade.     

Sequence analysis of the turkey coronavirus nucleocapsid protein gene and 3' untranslated region identifies the virus as a close relative of infectious bronchitis virus

The 3' end of the turkey coronavirus (TCV) genome (1740 bases) including the nucleocapsid (N) gene and 3' untranslated region (UTR) were sequenced and compared with published sequences of other avian and mammalian coronaviruses. The deduced sequence of the TCV N protein was determined to be 409 amino acids with a molecular mass of approximately 45 kDa. The TCV N protein was identical in size and had greater than 90% amino acid identity with published N protein sequences of infectious bronchitis virus (IBV); less than 21% identity was observed with N proteins of bovine coronavirus and transmissible gastroenteritis virus. The 3' UTR showed some variation among the three TCV strains examined, with two TCV strains, Minnesota and Indiana, containing 153 base segments which are not present in the NC95 strain. Nucleotide sequence identity between the 3' UTRs of TCV and IBV was greater than 78%. Similarities in both size and sequence of TCV and IBV N proteins and 3' UTRs provide additional evidence that these avian coronaviruses are closely related.     

Identification of two new polypeptides encoded by mRNA5 of the coronavirus infectious bronchitis virus.

The second smallest subgenomic messenger RNA, mRNA5, of the coronavirus infectious bronchitis virus includes in its "5' unique region" two separate open reading frames (5a and 5b), whose coding function has not so far been established, and thus it may represent a dicistronic messenger RNA. We report here that two polypeptides with the sizes expected for the 5a and 5b products can be synthesised by in vitro translation of a single artificial mRNA containing both the 5a and 5b ORFs. To establish whether these polypeptides represent genuine virus gene products, both the 5a and 5b coding sequences were expressed as bacterial fusion proteins, and these were used to raise monospecific antisera. Antisera raised against both the 5a and 5b-specific sequences recognized specifically proteins of the expected size in infectious bronchitis virus-infected chicken kidney and Vero cells, indicating that 5a and 5b do represent genuine virus genes, and suggesting that mRNA5 is indeed functionally dicistronic.     

A polycistronic mRNA specified by the coronavirus infectious bronchitis virus.

The third largest of the nested set of subgenomic mRNAs (mRNA3) from the coronavirus infectious bronchitis virus (IBV) contains three separate open reading frames (3a, 3b, and 3c) which are not present on the next smallest of the mRNAs, suggesting that this mRNA may be functionally polycistronic. However, although a protein product has been identified from the 3c open reading frame, to date the coding function of 3a and 3b has not been established. We present nucleotide sequence data suggesting that each of the three open reading frames is conserved in a variety of different IBV strains and further show, through the preparation of monospecific antisera against bacterial fusion proteins, that IBV-infected cells contain small amounts of the products of these ORFs. In vitro translation studies using synthetic mRNAs containing the 3a, 3b, and 3c open reading frames suggest strongly that all three proteins can be translated from a single molecular species, and expression studies carried out in intact cells support this conclusion. Thus mRNA3 of IBV appears to be functionally tricistronic.     

The genome sequence of lettuce necrotic stunt virus indicates a close relationship to Moroccan pepper virus

Lettuce necrotic stunt virus (LNSV) causes severe losses to lettuce production in the western United States, which results in stunting, necrosis and death on all non-crisphead lettuces, as well as flower abortion and yield losses in greenhouse tomato production. The genome of LNSV was sequenced and has an organization typical of viruses of the genus Tombusvirus. Sequence comparisons indicated that much of the genome is relatively closely related to tomato bushy stunt virus; however, the coat protein is very closely related to that of isolates of Moroccan pepper virus (MPV).     

Lithium chloride inhibits the coronavirus infectious bronchitis virus in cell culture.

The avian coronavirus infectious bronchitis virus (IBV) is a major economic pathogen of domestic poultry that, despite vaccination, causes mortality and significant losses in production. During replication of the RNA genome there is a high frequency of mutation and recombination, which has given rise to many strains of IBV and results in the potential for new and emerging strains. Currently the live-attenuated vaccine gives poor cross-strain immunity. Effective antiviral agents may therefore be advantageous in the treatment of IBV. Lithium chloride (LiCl) is a potent inhibitor of the DNA virus herpes simplex virus but not RNA viruses. The effect of LiCl on the replication of IBV was examined in cell culture using two model cell types; Vero cells, an African Green monkey kidney-derived epithelial cell line; and DF-1 cells, an immortalized chicken embryo fibroblast cell line. When treated with a range of LiCl concentrations, IBV RNA and protein levels and viral progeny production were reduced in a dose-dependent manner in both cell types, and the data indicated that inhibition was a cellular rather than a virucidal effect. Host cell protein synthesis still took place in LiCl-treated cells and the level of a standard cellular housekeeping protein remained unchanged, indicating that the effect of LiCl was specifically against IBV.     

Towards construction of viral vectors based on avian coronavirus infectious bronchitis virus for gene delivery and vaccine development

Manipulation of the coronavirus genome to accommodate and express foreign genes is an attractive approach for gene delivery and vaccine development. By using an infectious cloning system developed recently for the avian coronavirus infectious bronchitis virus (IBV), the enhanced green fluorescent protein (EGFP) gene, the firefly luciferase gene and several host and viral genes (eIF3f, SARS ORF6, Dengue virus 1 core protein gene) were inserted into various positions of the IBV genome, and the effects on gene expression, virus recovery, and stability in cell culture were studied. Selected viruses were also inoculated into chicken embryos for studies of foreign gene expression at different tissue level. The results demonstrated the stability of recombinant viruses depends on the intrinsic properties of the foreign gene itself as well as the position at which the foreign genes were inserted. For unstable viruses, the loss of expression of the inserted genes was found to result from a large deletion of the inserted gene and even IBV backbone sequences. This represents a promising system for development of coronavirus-based gene delivery vectors and vaccines against coronavirus and other viral infections in chicken.     

Generation of a recombinant avian coronavirus infectious bronchitis virus using transient dominant selection

A reverse genetics system for the avian coronavirus infectious bronchitis virus (IBV) has been described in which a full-length cDNA, corresponding to the IBV (Beaudette-CK) genome, was inserted into the vaccinia virus genome following in vitro assembly of three contiguous cDNAs [Casais, R., Thiel, V., Siddell, S.G., Cavanagh, D., Britton, P., 2001. Reverse genetics system for the avian coronavirus infectious bronchitis virus. J. Virol. 75, 12359-12369]. The method has subsequently been used to generate a recombinant IBV expressing a chimaeric S gene [Casais, R., Dove, B., Cavanagh, D., Britton, P., 2003. Recombinant avian infectious bronchitis virus expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell tropism. J. Virol. 77, 9084-9089]. Use of vaccinia virus as a vector for the full-length cDNA of the IBV genome has the advantage that modifications can be made to the IBV cDNA using homologous recombination, a method frequently used to insert and delete sequences from the vaccinia virus genome. We describe the use of homologous recombination as a method for modifying the Beaudette full-length cDNA, within the vaccinia virus genome, without the requirement for in vitro assembly of the IBV cDNA. To demonstrate the feasibility of the method we exchanged the ectodomain of the Beaudette spike gene for the corresponding region from IBV M41 and generated two recombinant infectious bronchitis viruses (rIBVs) expressing the chimaeric S protein, validating the method as an alternative way for generating rIBVs.     

Serological relationship among ten strains of avian infectious bronchitis virus

Ten strains of avian infectious bronchitis virus (IBV) were studied serologically by cross-neutralization test using rabbit and chicken immune sera. With the chicken sera all 10 IBV strains were antigenically related. In particular, anti-KH serum neutralized all heterologous strains except of the Ishida strain; Nerima strain was neutralized by all antisera except of anti-Ishida serum. Most cross-reactions were less or more heterologous, thus all 10 IBV strains seemed to belong to one serological type. Using rabbit sera, all strains except of Connecticut A-5968, cross-reacted with certain other strains. Most cross-reactions were partially heterologous showing one-way-relationship; heterologous relations were observed less frequently than with chicken sera.     

Genomic characterization of a proventriculitis-associated infectious bronchitis coronavirus

Transmissible proventriculitis associated with infectious bronchitis virus (IBV) was at first seen in eastern China in mid-1995, and is now endemic in China. Herein, the complete genome sequence of a proventiculitis-associated infectious bronchitis coronavirus (ZJ971) was sequenced and analyzed. Compared with the genome of the vaccine strain H120, ZJ971 had 54 nucleotide substitutions and a deletion in the 3′-UTR. The substitutions were in the regions of nsp2–nsp5, nsp7, nsp12, nsp13, nsp15, S and N genes, and the untranslating region. The results indicated that ZJ971 could be a variant of IBV strain H120.     

Activity of a purified His-tagged 3C-like proteinase from the coronavirus infectious bronchitis virus

Previous studies in vitro of the processing of cloned polyprotein fragments from the coronavirus infectious bronchitis virus (IBV) large open reading frame (ORF1), confirmed the activity of a predicted 3C-like proteinase (3CLP) domain and suggested that the proteinase is released autocatalytically from the polyprotein in the form of a 35 kDa protein, 3CLpro, capable of further cleavages in trans. In order to identify such cleavages within the ORF1 polyprotein mediated by 3CLpro, the proteinase was expressed in bacteria, purified and used in trans cleavage assays with polyprotein fragments lacking the 3CLP domain as targets. The proteinase was expressed as a polyprotein fragment which was able to process during expression in bacterial cells, releasing mature 3CLpro. A histidine (His₆) tag was introduced close to the C-terminus of the proteinase to aid purification. Processing demonstrated by the tagged proteinase was indistinguishable from that of the wild-type enzyme indicating that the site chosen for the tag was permissive. From these studies we were able to demonstrate trans cleavages consistent with the use of most of the previously predicted or identified sites within the open reading frame of gene 1. This tentatively completes the processing map for the ORF1 region with respect to 3CLpro.     

Detection of infectious bronchitis virus

The detection methods for infectious bronchitis virus (IBV) are reviewed. Advantages and disadvantages of available techniques of IBV detection by virus isolation, antigen or genome detection, and serology are discussed. Factors of influence on the level of success in detection of IBV after a disease outbreak are discussed, as are the possibilities and dangers of strain classification by protectotyping, serotyping, epitope-typing and genotyping.     

Role of phosphorylation clusters in the biology of the coronavirus infectious bronchitis virus nucleocapsid protein

The coronavirus infectious bronchitis virus (IBV) nucleocapsid (N) protein is an RNA binding protein which is phosphorylated at two conserved clusters. Kinetic analysis of RNA binding indicated that the C-terminal phosphorylation cluster was involved in the recognition of viral RNA from non-viral RNA. The IBV N protein has been found to be essential for the successful recovery of IBV using reverse genetics systems. Rescue experiments indicated that phosphorylated N protein recovered infectious IBV more efficiently when compared to modified N proteins either partially or non-phosphorylated. Our data indicate that the phosphorylated form of the IBV N protein plays a role in virus biology.