Infectious Bronchitis Virus Spike Research Articles

Study on the transient expression of infectious bronchitis virus spike protein in Nicotiana benthamiana leaves

Infectious bronchitis virus (IBV) is considered as one of the main causes of economic loss in chicken farms worldwide, especially in poultry producing nations. This virus, a member of the Coronaviridae family, is classified into different genotypes based on its surface spike glycoproteins which also play important roles in cell attachment and immune response. Due to continual genetic mutation of IBV, attenuated and inactivated vaccines show decreased effectiveness or lack of cross – protection; thus ongoing studies focus on the development of new generation vaccines to prevent new IBV outbreaks. This paper describes our study on expression of the antigenic region in S1 subunit of the spike in Nicotiana benthamiana. Two expression vectors carrying either S1 or receptor binding domain (RBD) coding gene (pCB301-S1 and pCB301-RBD) were constructed and transformed into tobacco leaves by agroinfiltration method. The RBD showed a clearly higher level of expression compared to the whole S1. Purification of RBD by immobilized metal ion chromatography and size exclusion chromatography represented a mixture of monomer, dimer and trimer glycoprotein with expected sizes in Western blot. In summary, this study demonstrated our primary success in establishing an expression model that could be used to investigate plant-based IBV recombinant vaccine.

Revealing Novel-Strain-Specific and Shared Epitopes of Infectious Bronchitis Virus Spike Glycoprotein Using Chemical Linkage of Peptides onto Scaffolds Precision Epitope Mapping.

The avian coronavirus, infectious bronchitis virus (IBV), is an economically important infectious disease affecting chickens, with a diverse range of serotypes found globally. The major surface protein, spike (S), has high diversity between serotypes, and amino acid differences in the S1 sub-unit are thought to be responsible for poor cross-protection afforded by vaccination. Here, we attempt to address this, by using epitope mapping technology to identify shared and serotype-specific immunogenic epitopes of the S glycoprotein of three major circulating strains of IBV, M41, QX, and 4/91, via CLIPS peptide arrays based on peptides from the S1 sub-units. The arrays were screened with sera from chickens immunised with recombinant IBV, based on Beau-R backbone expressing heterologous S, generated in two independent vaccination/challenge trials. The screening of sera from rIBV vaccination experiments led to the identification of 52 immunogenic epitopes on the S1 of M41, QX, and 4/91. The epitopes were assigned into six overlapping epitope binding regions. Based on accessibility and location in the hypervariable regions of S, three sequences, 25YVYYYQSAFRPPNGWHLQGGAYAVVNSTN54, 67TVGVIKDVYNQSVASI82, and 83AMTVPPAGMSWSVS96, were selected for further investigation, and synthetic peptide mimics were recognised by polyclonal sera. These epitopes may have the potential to contribute towards a broader cross-protective IBV vaccine.

Identification and Comparison of the Sialic Acid-Binding Domain Characteristics of Avian Coronavirus Infectious Bronchitis Virus Spike Protein.

Infectious bronchitis virus (IBV) infections are initiated by the transmembrane spike (S) glycoprotein, which binds to host factors and fuses the viral and cell membranes. The N-terminal domain of the S1 subunit of IBV S protein binds to sialic acids, but the precise location of the sialic acid binding domain (SABD) and the role of the SABD in IBV-infected chickens remain unclear. Here, we identify the S1 N-terminal amino acid (aa) residues 19 to 227 (209 aa total) of IBV strains SD (GI-19) and GD (GI-7), and the corresponding region of M41 (GI-1), as the minimal SABD using truncated protein histochemistry and neuraminidase assays. Both α-2,3- and α-2,6-linked sialic acids on the surfaces of CEK cells can be used as attachment receptors by IBV, leading to increased infection efficiency. However, 9-O acetylation of the sialic acid glycerol side chain inhibits IBV S1 and SABD protein binding. We further constructed recombinant strains in which the S1 gene or the SABD in the GD and SD genomes were replaced with the corresponding region from M41 by reverse genetics. Infecting chickens with these viruses revealed that the virulence and nephrotropism of rSDM41-S1, rSDM41-206, rGDM41-S1, and rGDM41-206 strains were decreased to various degrees compared to their parental strains. A positive sera cross-neutralization test showed that the serotypes were changed for the recombinant viruses. Our results provide insight into IBV infection of host cells that may aid vaccine design. IMPORTANCE To date, only α-2,3-linked sialic acid has been identified as a potential host binding receptor for IBV. Here, we show the minimum region constituting the sialic acid binding domain (SABD) and the binding characteristics of the S1 subunit of spike (S) protein of IBV strains SD (GI-19), GD (GI-7), and M41 (GI-1) to various sialic acids. The 9-O acetylation modification partially inhibits IBV from binding to sialic acid, while the virus can also bind to sialic acid molecules linked to host cells through an α-2,6 linkage, serving as another receptor determinant. Substitution of the putative SABD from strain M41 into strains SD and GD resulted in reduced virulence, nephrotropism, and a serotype switch. These findings suggest that sialic acid binding has diversified during the evolution of γ-coronaviruses, impacting the biological properties of IBV strains. Our results offer insight into the mechanisms by which IBV invades host cells.

A Peptide-Based Enzyme-Linked Immunosorbent Assay for Detecting Antibodies Against Avian Infectious Bronchitis Virus.

Infectious bronchitis virus (IBV) causes substantial loss to the poultry industry despite extensive vaccination. Assessing the antibody response is important for the development and evaluation of effective vaccines. We have developed an enzyme-linked immunosorbent assay (ELISA) for the detection of IBV-specific antibodies, using a synthetic peptide based on a conserved sequence in the IBV spike protein. This peptide-based ELISA (pELISA) specifically detects antibodies to different genotypes of IBV but not antibodies against other common chicken viruses. This assay could detect IBV-specific antibody response on as early as day 7 postinfection. In the testing with field serum samples collected from chickens administered with IBV vaccines, the sensitivity, specificity, and accuracy of pELISA were 98.30, 94.12, and 98.8%, respectively, relative to indirect immunofluorescence assay. Our data demonstrate that the pELISA is of value for the detection of IBV antibody and the evaluation of IBV vaccines.

The V617I Substitution in Avian Coronavirus IBV Spike Protein Plays a Crucial Role in Adaptation to Primary Chicken Kidney Cells.

The naturally isolated avian coronavirus infectious bronchitis virus (IBV) generally cannot replicate in chicken kidney (CK) cells. To explore the molecular mechanism of IBV adapting to CK cells, a series of recombinant viruses were constructed by chimerizing the S genes of CK cell-adapted strain H120 and non-adapted strain IBYZ. The results showed that the S2 subunit determines the difference in cell tropism of the two strains. After comparing the amino acid sequences of S protein of CK cell-adapted strain YZ120, with its parental strain IBYZ, three amino acid substitutions, A138V, L581F, and V617I, were identified. Using YZ120 as the backbone, one or more of the above-mentioned substitutions were eliminated to verify the correlation between these sites and CK cell tropism. The results showed that the CK cell tropism of the YZ120 strain depends on the V617I substitution, the change of L581F promoted the adaptation in CK cells, and the change at 138 position was not directly related to the CK cell tropism. Further validation experiments also showed that V617I had a decisive role in the adaptation of IBV to CK cells, but other areas of the virus genome also affected the replication efficiency of the virus in CK cells.

Vaccine design of coronavirus spike (S) glycoprotein in chicken: immunoinformatics and computational approaches

BackgroundInfectious bronchitis (IB) is a highly contagious respiratory disease in chickens and produces economic loss within the poultry industry. This disease is caused by a single stranded RNA virus belonging to Cronaviridae family. This study aimed to design a potential multi-epitopes vaccine against infectious bronchitis virus spike protein (S). Protein characterization was also performed for IBV spike protein.MethodsThe present study used various tools in Immune Epitope Database (IEDB) to predict conserved B and T cell epitopes against IBV spike (S) protein that may perform a significant role in provoking the resistance response to IBV infection.ResultsIn B cell prediction methods, three epitopes (1139KKSSYY1144,1140KSSYYT1145,1141SSYYT1145) were selected as surface, linear and antigenic epitopes.Many MHCI and MHCII epitopes were predicted for IBV S protein. Among them 982YYITARDMY990 and 983YITARDMYM991 epitopes displayed high antigenicity, no allergenicity and no toxicity as well as great linkage with MHCI and MHCII alleles. Moreover, docking analysis of MHCI epitopes produced strong binding affinity with BF2 alleles.ConclusionFive conserved epitopes were expected from spike glycoprotein of IBV as the best B and T cell epitopes due to high antigenicity, no allergenicity and no toxicity. In addition, MHC epitopes showed great linkage with MHC alleles as well as strong interaction with BF2 alleles. These epitopes should be designed and incorporated and then tested as multi-epitope vaccine against IBV.

Heat Shock Protein Member 8 Is an Attachment Factor for Infectious Bronchitis Virus.

Although infectious bronchitis virus (IBV) is the first coronavirus identified, little is known about which membrane protein of host cells could interact with IBV spike protein and facilitate the infection by the virus. In this study, by using a monoclonal antibody to the S1 protein of IBV M41 strain, we found that heat shock protein member 8 (HSPA8) could interact with spike protein of IBV. HSPA8 was found to be present on the cell membrane and chicken tissues, with highest expression level in the kidney. Results of co-IP and GST-pull-down assays indicated that the receptor binding domain (RBD) of IBV M41 could interact with HSPA8. The results of binding blocking assay and infection inhibition assay showed that recombinant protein HSPA8 and antibody to HSPA8 could inhibit IBV M41 infection of chicken embryonic kidney (CEK) cells. Further, we found that HSPA8 interacted with the N-terminal 19–272 amino acids of S1 of IBV Beaudette, H120 and QX strains and HSPA8 from human and pig also interacted with IBV M41-RBD. Finally the results of binding blocking assay and infection inhibition assay showed that recombinant HSPA8 protein and antibody to HSPA8 could inhibit IBV Beaudette strain infection of Vero cells that were treated with heparanase to remove heparan sulfate from the cell surface. Taken together, our results indicate that HSPA8 is a novel host factor involved in IBV infection.

The Infectious Bronchitis Coronavirus Envelope Protein Alters Golgi pH To Protect the Spike Protein and Promote the Release of Infectious Virus.

Coronaviruses (CoVs) assemble by budding into the lumen of the early Golgi complex prior to exocytosis. The small CoV envelope (E) protein plays roles in assembly, virion release, and pathogenesis. CoV E has a single hydrophobic domain (HD), is targeted to Golgi membranes, and has cation channel activity in vitro The E protein from avian infectious bronchitis virus (IBV) has dramatic effects on the secretory system, which require residues in the HD. Mutation of the HD of IBV E in a recombinant virus background results in impaired growth kinetics, impaired release of infectious virions, accumulation of IBV spike (S) protein on the plasma membrane compared to wild-type (WT) IBV-infected cells, and aberrant cleavage of IBV S on virions. We previously reported the formation of two distinct oligomeric pools of IBV E in transfected and infected cells. Disruption of the secretory pathway by IBV E correlates with a form that is likely monomeric, suggesting that the effects on the secretory pathway are independent of E ion channel activity. Here, we present evidence suggesting that the monomeric form of IBV E correlates with an increased Golgi luminal pH. Infection with IBV or expression of IBV E induces neutralization of Golgi pH, promoting a model in which IBV E alters the secretory pathway through interaction with host cell factors, protecting IBV S from premature cleavage and leading to the efficient release of infectious virus from the cells. This is the first demonstration of a coronavirus-induced alteration in the microenvironment of the secretory pathway.IMPORTANCE Coronaviruses are important human pathogens with significant zoonotic potential. Progress has been made toward identifying potential vaccine candidates for highly pathogenic human CoVs, including the use of attenuated viruses that lack the CoV E protein or express E mutants. However, no approved vaccines or antiviral therapeutics exist. Understanding the role of the CoV E protein in virus assembly and release is thus an important prerequisite for potential vaccines as well as in identifying novel antiviral therapeutics.

A self-adjuvanted nanoparticle based vaccine against infectious bronchitis virus.

Infectious bronchitis virus (IBV) affects poultry respiratory, renal and reproductive systems. Currently the efficacy of available live attenuated or killed vaccines against IBV has been challenged. We designed a novel IBV vaccine alternative using a highly innovative platform called Self-Assembling Protein Nanoparticle (SAPN). In this vaccine, B cell epitopes derived from the second heptad repeat (HR2) region of IBV spike proteins were repetitively presented in its native trimeric conformation. In addition, flagellin was co-displayed in the SAPN to achieve a self-adjuvanted effect. Three groups of chickens were immunized at four weeks of age with the vaccine prototype, IBV-Flagellin-SAPN, a negative-control construct Flagellin-SAPN or a buffer control. The immunized chickens were challenged with 5x104.7 EID50 IBV M41 strain. High antibody responses were detected in chickens immunized with IBV-Flagellin-SAPN. In ex vivo proliferation tests, peripheral mononuclear cells (PBMCs) derived from IBV-Flagellin-SAPN immunized chickens had a significantly higher stimulation index than that of PBMCs from chickens receiving Flagellin-SAPN. Chickens immunized with IBV-Flagellin-SAPN had a significant reduction of tracheal virus shedding and lesser tracheal lesion scores than did negative control chickens. The data demonstrated that the IBV-Flagellin-SAPN holds promise as a vaccine for IBV.

Oral immunization with a novel attenuated Salmonella Gallinarum encoding infectious bronchitis virus spike protein induces protective immune responses against fowl typhoid and infectious bronchitis in chickens

Fowl typhoid (FT), a septicemic disease caused by Salmonella Gallinarum (SG), and infectious bronchitis (IB) are two economically important avian diseases that affect poultry industry worldwide. Herein, we exploited a live attenuated SG mutant, JOL967, to deliver spike (S) protein 1 of IB virus (V) to elicit protective immunity against both FT and IB in chickens. The codon optimized S1 nucleotide sequence was cloned in-frame into a prokaryotic constitutive expression vector, pJHL65. Subsequently, empty pJHL65 or recombinant pJHL65-S1 plasmid was electroporated into JOL967 and the resultant clones were designated as JOL2068 and JOL2077, respectively. Our results demonstrated that the chickens vaccinated once orally with JOL2077 elicited significantly (p < 0.05) higher IBV-specific humoral and cell-mediated immunity compared to JOL2068 and PBS control groups. Consequently, on challenge with the virulent IBV strain at 28th day post-vaccination, JOL2077 vaccinated birds displayed significantly (p < 0.05) lower inflammatory lesions in virus-targeted tissues compared to control groups. Furthermore, 33.3% (2 of 6) of birds vaccinated with JOL2077 vaccine had shown virus recovery from tracheal tissues compared to 100% (6 of 6) recovery obtained in both the control groups. Against wild-type SG lethal challenge, both JOL2077 and JOL2068 vaccinated groups exhibited only 10% mortality compared to 80% mortality observed in PBS control group. In conclusion, we show that JOL2077 can induce efficient IBV- and carrier-specific protective immunity and can act as a bivalent vaccine against FT and IB. Further studies are warranted to investigate the potential of JOL2077 vaccine in broiler and young layer birds.

Identification of Novel Linear Epitopes Located in the Infectious Bronchitis Virus Spike S2 Region.

We identified novel linear epitopes on the infectious bronchitis virus (IBV) spike S2 region. The conformational structure of the IBV spike protein was predicted from a homologous protein, human coronavirus NL63 spike. Although the obtained structure was incomplete, most of the IBV spike protein structure was predicted; the N-terminus of the S1 region could not be predicted due to its variability. In the model, the region located in the proximity of the fusion peptide appeared to be well conserved, and we evaluated the antigenicity of these domains, which are involved in the membrane fusion machinery. Western blotting revealed that IBV TM86 spike residues 686-723 were antigenic. Epitope mapping analysis using synthesized peptides revealed that IBV TM86 spike 669-685 (SNFSTGAFNISLLLTPP), 686-697 (SNPRGRSFIEDL), and 692-703 (SFIEDLLFTSVE) residues were major linear epitopes; two identified epitopes (686-697 and 692-703) were covered by the fusion peptide, and the other epitope (669-685) was adjacent to the fusion peptide. Although the identified epitopes are identically located as the neutralizing epitope in severe acute respiratory syndrome coronavirus, the recombinant protein that includes those epitopes could not elicit neutralizing antibodies against IBV. This is the first report describing IBV spike S2 epitopes located in the proximity of the fusion peptide, and it is suggested that the spike fusion machinery of IBV may differ from that of severe acute respiratory syndrome coronavirus, or, alternatively, IBV may have another mechanism to penetrate the cell membrane.

A reverse genetics system for avian coronavirus infectious bronchitis virus based on targeted RNA recombination

BackgroundAvian coronavirus infectious bronchitis virus (IBV) is a respiratory pathogen of chickens that causes severe economic losses in the poultry industry worldwide. Major advances in the study of the molecular biology of IBV have resulted from the development of reverse genetics systems for the highly attenuated, cell culture-adapted, IBV strain Beaudette. However, most IBV strains, amongst them virulent field isolates, can only be propagated in embryonated chicken eggs, and not in continuous cell lines.MethodsWe established a reverse genetics system for the IBV strain H52, based on targeted RNA recombination in a two-step process. First, a genomic and a chimeric synthetic, modified IBV RNA were co-transfected into non-susceptible cells to generate a recombinant chimeric murinized (m) IBV intermediate (mIBV). Herein, the genomic part coding for the spike glycoprotein ectodomain was replaced by that of the coronavirus mouse hepatitis virus (MHV), allowing for the selection and propagation of recombinant mIBV in murine cells. In the second step, mIBV was used as the recipient. To this end a recombination with synthetic RNA comprising the 3′-end of the IBV genome was performed by introducing the complete IBV spike gene, allowing for the rescue and selection of candidate recombinants in embryonated chicken eggs.ResultsTargeted RNA recombination allowed for the modification of the 3′-end of the IBV genome, encoding all structural and accessory genes. A wild-type recombinant IBV was constructed, containing several synonymous marker mutations. The in ovo growth kinetics and in vivo characteristics of the recombinant virus were similar to those of the parental IBV strain H52.ConclusionsTargeted RNA recombination allows for the generation of recombinant IBV strains that are not able to infect and propagate in continuous cell lines. The ability to introduce specific mutations holds promise for the development of rationally designed live-attenuated IBV vaccines and for studies into the biology of IBV in general.

Gamma and Deltacoronaviruses in quail and pheasants from Northern Italy

In view of the restricted knowledge on the diversity of coronaviruses in poultry other than chicken, this study aimed to investigate the genetic diversity of coronaviruses in quail, pheasant, and partridge from two regions of Northern Italy. To this end, pools of tracheal and cloacal swabs from European quail (Coturnix Coturnix) and intestinal tract from pheasants (Phasianus Colchicus) and partridge (Perdix Perdix) flocks, with or without enteric signs, were collected during 2015. Avian coronavirus (Gammacoronavirus) was detected in quail not vaccinated against Infectious Bronchitis Virus (IBV) and in pheasants vaccinated with an IBV Massachusetts serotype. Based on DNA sequences for the gene encoding the S protein, the avian coronaviruses detected in the quail and pheasant are related to the IBV 793B and Massachusetts types, respectively. However, RNA-dependent RNA polymerase (RdRp) analyses showed the susceptibility of quail also to Deltacoronaviruses, suggesting that quail and pheasant avian coronaviruses share spike genes identical to chicken IBV spike genes and quail might host Deltacoronavirus.

Polymorphisms in the S1 spike glycoprotein of Arkansas-type infectious bronchitis virus (IBV) show differential binding to host tissues and altered antigenicity

Sequencing avian infectious bronchitis virus spike genes re-isolated from vaccinated chicks revealed that many sequence changes are found on the S1 spike gene. In the ArkDPI strain, Y43H and ∆344 are the two most common changes observed. This study aims to examine the roles of Y43H and ∆344 in selection in vivo. Using recombinant ArkDPI S1 proteins, we conducted binding assays on chicken tracheas and embryonic chorioallantoic membrane (CAM). Protein histochemistry showed that the Y43H change allows for enhanced binding to trachea, whereas the ArkDPI S1 spike with H43 alone was able to bind CAM. Using Western blot under denaturing conditions, ArkDPI serotype-specific sera did not bind to S1 proteins with ∆344, suggesting that ∆344 alters antigenicity of S1. These findings are important because they propose that specific changes in S1 enhances virus fitness by more effective binding to host tissues (Y43H) and by evading a vaccine-induced antibody response (∆344).

Genomic and single nucleotide polymorphism analysis of infectious bronchitis coronavirus

Infectious bronchitis virus (IBV) is a Gammacoronavirus that causes a highly contagious respiratory disease in chickens. A QX-like strain was analysed by high-throughput Illumina sequencing and genetic variation across the entire viral genome was explored at the sub-consensus level by single nucleotide polymorphism (SNP) analysis. Thirteen open reading frames (ORFs) in the order 5′-UTR-1a-1ab-S-3a-3b-E-M-4b-4c-5a-5b-N-6b-3′UTR were predicted. The relative frequencies of missense: silent SNPs were calculated to obtain a comparative measure of variability in specific genes. The most variable ORFs in descending order were E, 3b, 5′UTR, N, 1a, S, 1ab, M, 4c, 5a, 6b. The E and 3b protein products play key roles in coronavirus virulence, and RNA folding demonstrated that the mutations in the 5′UTR did not alter the predicted secondary structure. The frequency of SNPs in the Spike (S) protein ORF of 0.67% was below the genomic average of 0.76%. Only three SNPS were identified in the S1 subunit, none of which were located in hypervariable region (HVR) 1 or HVR2. The S2 subunit was considerably more variable containing 87% of the polymorphisms detected across the entire S protein. The S2 subunit also contained a previously unreported multi-A insertion site and a stretch of four consecutive mutated amino acids, which mapped to the stalk region of the spike protein. Template-based protein structure modelling produced the first theoretical model of the IBV spike monomer. Given the lack of diversity observed at the sub-consensus level, the tenet that the HVRs in the S1 subunit are very tolerant of amino acid changes produced by genetic drift is questioned.

Differences in the tissue tropism to chicken oviduct epithelial cells between avian coronavirus IBV strains QX and B1648 are not related to the sialic acid binding properties of their spike proteins.

The avian coronavirus (AvCoV) infectious bronchitis virus (IBV) is a major poultry pathogen. A characteristic feature of IBV is the occurrence of many different strains belonging to different serotypes, which makes a complete control of the disease by vaccinations a challenging task. Reasons for differences in the tissue tropism and pathogenicity between IBV strains, e.g. a predilection for the kidneys or the oviduct are still an open question. Strains of the QX genotype have been major pathogens in poultry flocks in Asia, Europe and other parts of the world. They are the cause of severe problems with kidney disease and reproductive tract disorders. We analysed infectivity and binding properties of the QX strain and compared them with those of the nephropathogenic strain B1648. As most IBV strains do not infect permanent cell lines and show infection only in primary chicken cells of the target organs, we developed a culture system for chicken oviduct explants. The epithelial cells of the oviduct showed a high susceptibility to infection by the QX strain and were almost resistant to infection by the nephropathogenic B1648 strain. Binding tests with isolated primary oviduct epithelial cells and soluble S1 proteins revealed that S1 proteins of two IBV strains bound with the same efficiency to oviduct epithelial cells. This attachment was sialic acid dependent, indicating that the sugar binding property of IBV spike proteins is not the limiting factor for differences in infection efficiency for the oviduct of the corresponding viruses.

Sialic Acid Binding Properties of Soluble Coronavirus Spike (S1) Proteins: Differences between Infectious Bronchitis Virus and Transmissible Gastroenteritis Virus

The spike proteins of a number of coronaviruses are able to bind to sialic acids present on the cell surface. The importance of this sialic acid binding ability during infection is, however, quite different. We compared the spike protein of transmissible gastroenteritis virus (TGEV) and the spike protein of infectious bronchitis virus (IBV). Whereas sialic acid is the only receptor determinant known so far for IBV, TGEV requires interaction with its receptor aminopeptidase N to initiate infection of cells. Binding tests with soluble spike proteins carrying an IgG Fc-tag revealed pronounced differences between these two viral proteins. Binding of the IBV spike protein to host cells was in all experiments sialic acid dependent, whereas the soluble TGEV spike showed binding to APN but had no detectable sialic acid binding activity. Our results underline the different ways in which binding to sialoglycoconjugates is mediated by coronavirus spike proteins.

Characterization of cellular furin content as a potential factor determining the susceptibility of cultured human and animal cells to coronavirus infectious bronchitis virus infection

In previous studies, the Beaudette strain of coronavirus infectious bronchitis virus (IBV) was adapted from chicken embryo to Vero, a monkey kidney cell line, by serial propagation for 65 passages. To characterize the susceptibility of other human and animal cells to IBV, 15 human and animal cell lines were infected with the Vero-adapted IBV and productive infection was observed in four human cell lines: H1299, HepG2, Hep3B and Huh7. In other cell lines, the virus cannot be propagated beyond passage 5. Interestingly, cellular furin abundance in five human cell lines was shown to be strongly correlated with productive IBV infection. Cleavage of IBV spike protein by furin may contribute to the productive IBV infection in these cells. The findings that IBV could productively infect multiple human and animal cells of diverse tissue and organ origins would provide a useful system for studying the pathogenesis of coronavirus.

Histopathological and Immunohistochemical Study of Air Sac Lesions Induced by Two Strains of Infectious Bronchitis Virus

Infectious bronchitis virus (IBV) is a highly contagious respiratory coronavirus of domestic chickens. Although mortality is low, infection with IBV results in substantial losses for the egg and meat chicken industries. Despite the economic importance of IBV and decades of research into the pathogenesis of infection, significant gaps in our knowledge exist. The aim of this study was to compare the early progression of air sac lesions in birds receiving a vaccine strain of the virus or a more virulent field strain. The air sacs are lined by different types of epithelia and are relatively isolated from the environment, so they represent a unique tissue in which to study virus-induced lesions. Both the pathogenic and vaccine strains of the virus produced significant lesions; however, the lesions progressed more rapidly in the birds receiving the pathogenic strain. Immunohistochemistry demonstrated that in birds infected with the pathogenic strain of virus, IBV spike protein is detected first in the ciliated cells lining the air sac. These preliminary data provide important clues regarding potential mechanisms for IBV tissue tropism and spread and show that the nature of the virus isolate influences the early progression of IBV infection.

Contribution of Trafficking Signals in the Cytoplasmic Tail of the Infectious Bronchitis Virus Spike Protein to Virus Infection

Coronavirus spike (S) proteins are responsible for binding and fusion with target cells and thus play an essential role in virus infection. Recently, we identified a dilysine endoplasmic reticulum (ER) retrieval signal and a tyrosine-based endocytosis signal in the cytoplasmic tail of the S protein of infectious bronchitis virus (IBV). Here, an infectious cDNA clone of IBV was used to address the importance of the S protein trafficking signals to virus infection. We constructed infectious cDNA clones lacking the ER retrieval signal, the endocytosis signal, or both. The virus lacking the ER retrieval signal was viable. However, this virus had a growth defect at late times postinfection and produced larger plaques than IBV. Further analysis confirmed that the mutant S protein trafficked though the secretory pathway faster than wild-type S protein. A more dramatic phenotype was obtained when the endocytosis signal was mutated. Recombinant viruses lacking the endocytosis signal (in combination with a mutated dilysine signal or alone) could not be recovered, even though transient syncytia were formed in transfected cells. Our results suggest that the endocytosis signal of IBV S is essential for productive virus infection.