Infectious Bronchitis Virus H120 Research Articles

Genetic and pathogenic characterization of new infectious bronchitis virus strains in the GVI-1 and GI-19 lineages isolated in central China

Avian infectious bronchitis (IB) is a highly contagious infectious disease caused by infectious bronchitis virus (IBV), which is prevalent in many countries worldwide and causes serious harm to the poultry industry. At present, many commercial IBV vaccines have been used for the prevention and control of IB; however, IB outbreaks occur frequently. In this study, two new strains of IBV, SX/2106 and SX/2204, were isolated from two flocks which were immunized with IBV H120 vaccine in central China. Phylogenetic and recombination analysis indicated that SX/2106, which was clustered into the GI-19 lineage, may be derived from recombination events of the GI-19 and GI-7 strains and the LDT3-A vaccine. Genetic analysis showed that SX/2204 belongs to the GVI-1 lineage, which may have originated from the recombination of the GI-13 and GVI-1 strains and the H120 vaccine. The virus cross-neutralization test showed that the antigenicity of SX/2106 and SX/2204 was different from H120. Animal experiments found that both SX/2106 and SX/2204 could replicate effectively in the lungs and kidneys of chickens and cause disease and death, and H120 immunization could not provide effective protection against the two IBV isolates. It is noteworthy that the pathogenicity of SX/2204 has significantly increased compared to the GVI-1 strains isolated previously, with a mortality rate up to 60%. Considering the continuous mutation and recombination of the IBV genome to produce new variant strains, it is important to continuously monitor epidemic strains and develop new vaccines for the prevention and control of IBV epidemics.

Replication and vaccine protection of multiple infectious bronchitis virus strains in pheasants (Phasianus colchicus).

This study demonstrates that infectious bronchitis virus (IBV) strain M41, which is pathogenic for chickens, is nonpathogenic for pheasants. However, M41 replicated in the respiratory tracts of most inoculated pheasants and the virus was shed from their respiratory tracts in the early stages of infection (4 and 8 dpc). Similarly, the attenuated IBV H120 vaccine strain also replicated and the virus was shed from their respiratory tracts of most inoculated pheasants, whereas the pheasant coronavirus (PhCoV) I0623/17 replicated in the respiratory tracts of all challenged pheasants, which then shed virus for a long period of time. Strain M41 also replicated in selected tissues of the inoculated pheasants, including the lung, kidney, proventriculus, and cecal tonsil, although the viral titers were very low. Therefore, it was important to establish whether the H120 vaccine, which has a limited replication capacity in pheasants, induces a protective immune response to both "homologous" M41 and "heterologous" I0623/17 challenge. Vaccination with H120 induced humoral responses, and the replication of M41 was reduced or restricted in the tissues of the H120-vaccinated pheasants compared with its replication in unvaccinated birds. This implies that partial protection was conferred on pheasants by vaccination with the H120 vaccine. Prolonged viral replication and a large number of birds shedding virus into the respiratory tract were also observed in the unvaccinated pheasants after inoculation with M41. However, only limited protection against challenge with PhCoV I0623/17 was conferred on pheasants vaccinated with H120, largely because the replication of H120 in pheasants was limited, thus, limiting the immune responses induced by it. The low amino acid identity of the S1 subunit of the S proteins of H120 and I0623/17 might also account, at least in part, for the poor cross-protective immunity induced by H120. These results suggest that further work is required to rationally design vaccines that confer effective protection against PhCoV infection in commercial pheasant stocks.

A label-free electrochemical assay for coronavirus IBV H120 strain quantification based on equivalent substitution effect and AuNPs-assisted signal amplification.

A label-free electrochemical strategy is proposed combining equivalent substitution effect with AuNPs-assisted signal amplification. According to the differences of S1 protein in various infectious bronchitis virus (IBV) strains, a target DNA sequence that can specifically recognize H120 RNA forming a DNA-RNA hybridized double-strand structure has been designed. Then, the residual single-stranded target DNA is hydrolyzed by S1 nuclease. Therefore, the content of target DNA becomes equal to the content of virus RNA. After equivalent coronavirus, the target DNA is separated from DNA-RNA hybridized double strand by heating, which can partly hybridize with probe 2 modified on the electrode surface and probe 1 on AuNPs’ surface. Thus, AuNPs are pulled to the surface of the electrode and the abundant DNA on AuNPs’ surface could adsorb a large amount of hexaammineruthenium (III) chloride (RuHex) molecules, which produce a remarkably amplified electrochemical response. The voltammetric signal of RuHex with a peak near − 0.28 V vs. Ag/AgCl is used as the signal output. The proposed method shows a detection range of 1.56e−9 to 1.56e−6 μM with the detection limit of 2.96e−10 μM for IBV H120 strain selective quantification detection, exhibiting good accuracy, stability, and simplicity, which shows a great potential for IBV detection in vaccine research and avian infectious bronchitis diagnosis. Graphical abstract Electronic supplementary materialThe online version of this article (10.1007/s00604-020-04582-3) contains supplementary material, which is available to authorized users.

Construction of an infectious bronchitis virus vaccine strain carrying chimeric S1 gene of a virulent isolate and its pathogenicity analysis.

Infectious bronchitis virus (IBV) is a member of genus gamma-coronavirus in the family Coronaviridae, causing serious economic losses to the poultry industry. Reverse genetics is a common technique to study the biological characteristics of viruses. So far, there is no BAC reverse genetic system available for rescue of IBV infectious clone. In the present study, a new strategy for the construction of IBV infectious cDNA clone was established. The full-length genomic cDNA of IBV vaccine strain H120 was constructed in pBAC vector from four IBV fragment subcloning vectors by homologous recombination, which contained the CMV promoter at the 5′ end and the hepatitis D virus ribozyme (HDVR) sequence and bovine growth hormone polyadenylation (BGH) sequence after the polyA tail at the 3′ end of the full-length cDNA. Subsequently, using the same technique, another plasmid pBAC-H120/SCS1 was also constructed, in which S1 gene from IBV H120 strain was replaced with that of a virulent SC021202 strain. Recombinant virus rH120 and rH120/SCS1 were rescued by transfecting the plasmids into BHK cells and passaged in embryonated chicken eggs. Finally, the pathogenicity of both the recombinant virus strains rH120 and rH120/SCS1 was evaluated in SPF chickens. The results showed that the chimeric rH120/SCS1 strain was not pathogenic compared with the wild-type IBV SC021202 strain and the chickens inoculated with rH120/SCS1 could resist challenge infection by IBV SC021202. Taken together, our results indicate that BAC reverse genetic system could be used to rescue IBV in vitro and IBV S1 protein alone might not be the key factor for IBV pathogenicity.Key points • BAC vector was used to construct IBV full-length cDNA by homologous recombination. • Based on four subcloning vectors, a recombinant chimeric IBV H120/SCS1 was constructed and rescued. • Pathogenicity of H120/SCS1 was similar to that of H120, but different to that of SC021202.

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.

Recombinant infectious bronchitis virus (IBV) H120 vaccine strain expressing the hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) protects chickens against IBV and NDV challenge.

Infectious bronchitis (IB) and Newcastle disease (ND) are common viral diseases of chickens, which are caused by infectious bronchitis virus (IBV) and Newcastle disease virus (NDV), respectively. Vaccination with live attenuated strains of IBV-H120 and NDV-LaSota are important for the control of IB and ND. However, conventional live attenuated vaccines are expensive and result in the inability to differentiate between infected and vaccinated chickens. Therefore, there is an urgent need to develop new efficacious vaccines. In this study, using a previously established reverse genetics system, we generated a recombinant IBV virus based on the IBV H120 vaccine strain expressing the haemagglutinin-neuraminidase (HN) protein of NDV. The recombinant virus, R-H120-HN/5a, exhibited growth dynamics, pathogenicity and viral titers that were similar to those of the parental IBV H120, but it had acquired hemagglutination activity from NDV. Vaccination of SPF chickens with the R-H120-HN/5a virus induced a humoral response at a level comparable to that of the LaSota/H120 commercial bivalent vaccine and provided significant protection against challenge with virulent IBV and NDV. In summary, the results of this study indicate that the IBV H120 strain could serve as an effective tool for designing vaccines against IB and other infectious diseases, and the generation of IBV R-H120-HN/5a provides a solid foundation for the development of an effective bivalent vaccine against IBV and NDV.

Molecular analysis of the nucleocapsid gene and 3' untranslated region of two infectious Bronchitis Virus field isolates from Iranian poultry farms

Infectious bronchitis (IB) is an economically important disease of chickens. Due to the emergence of new variants of infectious bronchitis virus (IBV), the control of IB has become a serious problem for the poultry industry worldwide. In the present study, the nucleocapsid gene (N) and 3' untranslated region (UTR) of two IBVs isolated from Iranian poultry farms were sequenced and compared with other IBV strains. Based on nucleotide identity, the N gene and 3' UTR sequences of Iranian IBVs showed 90% similarity to the commonly used IBV vaccine strains, H52 and H120. However, based on phylogenetic analyses, Iranian IBVs were found to cluster separately from the IBV vaccine strains used in Iran as well as other IBVs isolated in China, Australia and the United States. It was concluded that IBVs circulating in Iran are genetically distinct from IBV vaccine strains that have been used in Iran for many years. Therefore, it is important to develop a new vaccine based on these newly identified strains for controlling IB in Iranian poultry farms.

Molecular typing of N gene and 3′ untranslated region of IBV field isolates and vaccine strains using RT-PCR and RFLP

Whole nucleocapsid (N) gene and 3′ untranslated region (UTR) of infectious bronchitis virus (IBV) vaccines and Iranian field isolates were amplified using reverse transcription and polymerase chain reaction (RT-PCR). The amplified fragments were subjected to digestion using two restriction endonuclease enzymes, AluI and MnlI. Five different restriction fragment length polymorphism (RFLP) patterns were generated using both enzymes, classifying IBV strains into five similar groups. Based on RT-PCR and RFLP analysis of the N gene and 3′ UTR, IBV vaccine strains H52, H120 and MA5 all from the same serotype generated identical patterns using both enzymes. Vaccine strain and field isolate of 4/91 also had the same pattern distinct from other IBVs. The IB88 vaccine strain and two other field isolates MNS-7862-1 and Ur1/09 had three different RFLP patterns distinguishable from each other and the other IBVs. In conclusion, RT-PCR and RFLP analysis of the N gene and 3′ UTR could be employed as a useful method for differentiating IBV strains especially in cases where S1 gene amplification is not successful because of its highly variable nature among different IBVs.

Course of infection and immune responses in the respiratory tract of IBV infected broilers after superinfection with E. coli

Colibacillosis results from infection with avian pathogenic Escherichia coli bacteria. Healthy broilers are resistant to inhaled E. coli, but previous infection with vaccine or virulent strains of Infectious Bronchitis Virus (IBV) predisposes birds for severe colibacillosis. The aim of this study was to investigate how IBV affects the course of events upon infection with E. coli. Broilers were inoculated with IBV H120 vaccine virus or virulent M41 and challenged 5 days later with E. coli 506. A PBS and E. coli group without previous virus inoculation were included. Sections of trachea, lung and airsacs were stained for CD4, CD8, γδ-TCR, αβ1-TCR, and for macrophages (KUL-01) and both pathogens. Changes in the mucociliary barrier of trachea, lung and airsacs did not predispose for bacterial superinfection. The disease in the lungs of the E. coli group and both IBV/ E. coli groups was similar. Lesions in the airsacs were more pronounced and of longer duration in the IBV/ E. coli groups. The immunocytological changes differed substantially between the E. coli group and both IBV/ E. coli groups. In trachea, lungs and airsacs the CD4+ and CD8+ populations were significantly larger than in the E. coli and PBS groups. In the lungs and the airsacs the macrophages were more numerous in the IBV/ E. coli and the E. coli groups than in the PBS group. The presence of high numbers of T cells and macrophages in IBV infected birds most likely induced an altered immune response, which is responsible for the enhanced clinical signs of colibacillosis.

Transmissibility of Infectious Bronchitis Virus H120 Vaccine Strain Among Broilers Under Experimental Conditions

The aim of this study was to quantify transmission of infectious bronchitis virus (IBV) H120 vaccine strain among broilers, and to assess whether birds that have been exposed to vaccine strain-shedding birds were protected against clinical signs after infection with a virulent strain of the same serotype. A transmission experiment and a replicate were carried out, each with six groups of commercial broilers. At day of hatch (n = 30) or at 15 days of age (n = 20), half of each group was inoculated with either IBV H120 vaccine (H120 group), virulent IBV M41 (M41 group), or were mock-infected, thereby contact-exposing the other half of each group. Nasal discharge was recorded, and antibody response and virus shedding were measured. To measure clinical protection, four weeks after inoculation all birds, in all groups, were challenged with IBV M41. The reproduction ratio (R; the average number of contact infections caused by one infectious bird) was determined to quantify virus transmission. All contact-exposed birds, except for one in an H120 group, became infected with either IBV H120 or IBV M41. Almost all birds contact-infected with IBV H120 or IBV M41 were subsequently protected against clinical signs after challenge with IBV M41. The lower limits of the 95% confidence interval (CI) of the R of IBV H120 vaccine, and of IBV M41, were significantly <1. For both IBV H120 and IBV M41, the 95% CI was [2.1-infinity] following inoculation at day of hatch and [1.8-infinity] after inoculation at 15 days of age. This finding demonstrates that IBV H120 vaccine is able to spread extensively among broilers. This implies that this vaccine strain might be able to become endemically present in the poultry population. It also implies that, even if not all birds received vaccine during spray application, due to the ability of the vaccine to spread in the flock, they will most likely be protected against clinical signs after a subsequent field virus infection.

Transmissibility of Infectious Bronchitis Virus H120 Vaccine Strain among Broilers under Experimental Conditions

Transmissibility of Infectious Bronchitis Virus H120 Vaccine Strain among Broilers under Experimental Conditions

The role of phagocytic cells in enhanced susceptibility of broilers to colibacillosis after Infectious Bronchitis Virus infection

Colibacillosis results from infection with avian pathogenic Escherichia coli bacteria. Healthy broilers are resistant to inhaled E. coli, but previous infection with vaccine or virulent strains of Infectious Bronchitis Virus (IBV) predisposes birds for severe colibacillosis. We investigated whether IBV affects recruitment and function of phagocytic cells and examined NO production, phagocytic and bactericidal activity, and kinetics of peripheral blood mononuclear cells (PBMC) and splenocytes. Moreover, we measured cytokine mRNA expression in lung and spleen samples. Broilers were inoculated with IBV H120 vaccine or virulent M41 and challenged 5 days later with E. coli 506. A PBS control and E. coli group without previous virus inoculation were also included. Birds were sacrificed at various time points after inoculation (h/dpi). Inoculation with IBV induced extended and more severe colibacillosis than with E. coli alone. At 4 dpi, the number of KUL-01 + PBMC in all E. coli-inoculated groups was significantly higher than in PBS-inoculated birds, which correlated with lesion scores. From 1 to 4 dpi, NO production by PBMC from all E. coli-inoculated animals was elevated compared to PBS birds. Bactericidal activity of PBMC in IBV-inoculated animals at 7 dpi was lower than in PBS- and E. coli-inoculated birds, but phagocytic capacity and recruitment were not severely impaired. In spleen samples of IBV-infected animals reduced expression of IL-1β, IL-6, IL-8, IL-10, IL-18 and IFN-γ mRNA was found 1 dpi. Our results suggest that enhanced colibacillosis after IBV infection or vaccination is caused at least by altered innate immunity and less by impairment of phagocytic cell function.

Infection of HeLa cells by avian infectious bronchitis virus is dependent on cell status

To investigate the adaptation of avian infectious bronchitis virus (IBV) in a human cell line may be beneficial to understanding the potential mechanisms of coronavirus interspecies infection. The current study addressed the poor replication of IBV in the HeLa human cell line demonstrated in previous reports. We showed that IBV strains M41, H52, H120 and Gray could be propagated in HeLa cells with distinct cytopathic effect. The virus titre in freshly dispersed HeLa cells was 1000-fold higher than in cell monolayers. Trypsin was not the determinant for the viral replication, suggesting that the restriction of IBV replication in HeLa cells is the result of intracellular events rather than the binding to or fusion with host cells. These IBV strains replicated to an average titre of 103.4±0.2/0.1 ml median tissue culture infectious doses in freshly dispersed HeLa cells and maintained this titre for the first 12 passages. Then an approximately 10-fold increase (104.20±0.19/0.1 ml) occurred in passage 13, which was maintained to passage 16, after which there was another, bigger rise to 106.6±0.3/0.1 ml in passage 17. This titre was maintained until passage 24 when the experiment was terminated. The IBV M41 S1 gene was amplified and sequenced for passages 0, 5 and 21. There was only one amino acid replacement in the S1 protein, in passage 21. The presence of sialic acid on HeLa cells contributed to efficient virus replication, while human aminopeptidase N was not involved in the infection. Haemagglutinin activity gradually reduced with increased passages. These results indicated that the virus adaptation would probably be determined by host cell modification such as receptor glycosylation and different receptor utilization instead of viral gene mutation.

Transmission of infectious bronchitis virus within vaccinated and unvaccinated groups of chickens

The aim of this study was to determine whether vaccination against infectious bronchitis virus (IBV) reduces virus transmission, i.e. to test whether IBV transmission among vaccinated chickens is significantly reduced compared to that among unvaccinated chickens. In two vaccinated and two unvaccinated groups of SPF chickens, a standard measure for virus transmission, the reproduction ratio (R) was determined. R is defined as the average number of new infections caused by one typical infectious individual during its entire infectious period. A single vaccination by eye‐drop with IBV H120 reduced the transmission of the IBV challenge virus among the vaccinated chickens (estimated R = 0.69, s.e. = 0.33) significantly (P < 0.05) compared to the transmission among the unvaccinated chickens (estimated R = 19.95, s.e. = 12.41). The possible implications for further study, including selection or development of vaccines are discussed.