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.     

Rapid development and evaluation of a live-attenuated QX-like infectious bronchitis virus vaccine

Infectious bronchitis (IB) is an acute, highly contagious disease, which causes economic losses to the poultry industry worldwide. To control the disease, biosecurity and vaccination are required. In the current research, we rapidly attenuated a QX-like IBV field strain ZYY-2014 using passage in embryos at limiting dilution and tested the safety and efficacy of the attenuated Chinese QX-like IBV strain ZYYR-2014 in 1-day-old specific-pathogen-free (SPF) chickens through spray route. Our result revealed that the attenuated strain presented a decreased pathogenicity in 1-day-old chickens. The strain ZYY-2014 inoculated birds presented typical IBV clinical signs with a mortality of 43%, while the attenuated strain ZYYR-2014 inoculated birds remained healthy. The strain ZYYR-2014 also presented stronger antibody responses and lower viral loads in tracheas, lungs and kidneys. When vaccinated through spray route into 1-day-old SPF chickens, our data suggest a potential of the attenuated ZYYR-2014 strain as a vaccine candidate applied in hatchery, which can contribute in preventing the QX-like IBV infections. Furthermore, attenuation by passage at limiting dilution could be applied for rapid vaccine development against emerging strains.     

Comparative analysis of four Massachusetts type infectious bronchitis coronavirus genomes reveals a novel Massachusetts type strain and evidence of natural recombination in the genome

Four Massachusetts-type (Mass-type) strains of infectious bronchitis coronavirus (IBV) were compared genetically with the pathogenic M41 and H120 vaccine strains using the complete genomic sequences. The results revealed that strains ck/CH/LNM/091017 and ck/CH/LDL/101212 were closely related to the H120 vaccine, which suggests that they might represent re-isolations of vaccine strains or variants of vaccine strains that have resulted from the accumulated point mutations after several passages in chickens. In contrast, strains ck/CH/LHLJ/07VII and ck/CH/LHLJ/100902 had a close genetic relationship with the pathogenic M41 strain. In addition, molecular markers have been identified that distinguish between field and vaccine (or vaccine-like) Mass-type viruses, which may be able to differentiate between field and vaccine strains for diagnostic purposes. Phylogenetic analysis, and pairwise comparison of full-length genomes and the nine genes, identified the occurrence of recombination events in the genome of strain CK/VH/LHLJ/07VII, which suggests that this virus originated from recombination events between M41- and H120-like strains at the switch site located at the 3′ end of the nucleocapsid (N) genes. To our knowledge, this is the first time that evidence for the evolution and natural recombination under field conditions between Mass-type pathogenic and vaccinal IBV strains has been documented. These findings provide insights into the emergence and evolution of the Mass-type IB coronaviruses and may help to explain the emergence of Mass-type IBV in chicken flocks all over the world.     

Effects of infectious bronchitis virus vaccine on the oviduct of hens

In Australia, currently, all pullets reared for egg production are vaccinated with live attenuated strains of infectious bronchitis virus. Various vaccines and protocols to control this viral disease have been developed, although the severity of the disease varies from place to place and flock to flock. In the present trial, the effects of vaccine strains A3 and Vic S on the oviduct of laying hens were assessed by histopathology, electron microscopy, serology and also by determining the presence and persistence of viral RNA in the oviduct by real time PCR following the experimental infection. Birds were either unvaccinated or vaccinated with both A3 and Vic S and then mock-infected, challenged with the same attenuated strains, either A3 or Vic S. Some respiratory signs were observed, but were mild and short-lived. There was no drop in egg production in any of the groups. However, there was visual loss of shell colour in the unvaccinated hens challenged with the Vic S strain. Mild histopathology was recorded only in terms of lymphocyte infiltration and occasional submucosal oedema in the infundibulum and very mild gland dilatation in the magnum. Microscopic lesions were not recorded in the isthmus, tubular shell gland or shell gland pouch. Cilia loss was not observed in any region of the oviduct using scanning electron microscopy. Both the A3 and Vic S vaccine strains were detected in the oviduct of vaccinated and unvaccinated hens, mainly on the 12th day p.i. These results indicate that the A3 and Vic S strains replicate at a low level in the oviduct without causing significant damage and hence are safe for the oviduct.     

Sequence evidence for RNA recombination in field isolates of avian coronavirus infectious bronchitis virus

Under laboratory conditions coronaviruses were shown to have a high frequency of recombination. In The Netherlands, vaccination against infectious bronchitis virus (IBV) is performed with vaccines that contain several life-attenuated virus strains. These highly effective vaccines may create ideal conditions for recombination, and could therefore be dangerous in the long term. This paper addresses the question of the frequency of recombination of avian coronavirus IBV in the field. A method was sought to detect and quantify recombination from sequence data. Nucleotide sequences of eight IBV isolates in a region of the genome suspected to contain recombination, were aligned and compared. Phylogenetic trees were constructed for different sections of this region. Differences in topology between these trees were observed, suggesting that in three out of eight strains in vivo RNA recombinant had occurred.     

Recombinant live attenuated avian coronavirus vaccines with deletions in the accessory genes 3ab and/or 5ab protect against infectious bronchitis in chickens

Avian coronavirus infectious bronchitis virus (IBV) is a respiratory pathogen of chickens, causing severe economic losses in poultry industry worldwide. Live attenuated viruses are widely used in both the broiler and layer industry because of their efficacy and ability to be mass applied. Recently, we established a novel reverse genetics system based on targeted RNA recombination to manipulate the genome of IBV strain H52. Here we explore the possibilities to attenuate IBV in a rational way in order to generate safe and effective vaccines against virulent IBV (van Beurden et al., 2017). To this end, we deleted the nonessential group-specific accessory genes 3 and/or 5 in the IBV genome by targeted RNA recombination and selected the recombinant viruses in embryonated eggs. The resulting recombinant (r) rIBV-Δ3ab, rIBV-Δ5ab, and rIBV-Δ3ab5ab could be rescued and grew to the same virus titer as recombinant and wild type IBV strain H52. Thus, genes 3ab and 5ab are not essential for replication in ovo. When administered to one-day-old chickens, rIBV-Δ3ab, rIBV-Δ5ab, and rIBV-Δ3ab5ab showed reduced ciliostasis as compared to rIBV H52 and wild type H52, indicating that the accessory genes contribute to the pathogenicity of IBV. After homologous challenge with the virulent IBV strain M41, all vaccinated chickens were protected against disease based on reduced loss of ciliary movement in the trachea compared to the non-vaccinated but challenged controls. Taken together, deletion of accessory genes 3ab and/or 5ab in IBV resulted in mutant viruses with an attenuated phenotype and the ability to induce protection in chickens. Hence, targeted RNA recombination based on virulent IBV provides opportunities for the development of a next generation of rationally designed live attenuated IBV vaccines.     

Avian coronavirus infectious bronchitis attenuated live vaccines undergo selection of subpopulations and mutations following vaccination

In this study, we were interested in determining if high titered egg adapted modified live infectious bronchitis virus (IBV) vaccines contain spike gene related quasispecies that undergo selection in chickens, following vaccination. We sequenced the spike glycoprotein of 12 IBV vaccines (5 different serotypes from 3 different manufacturers) directly from the vaccine vial, then compared that sequence with reisolated viruses from vaccinated and contact-exposed birds over time. We found differences in the S1 sequence within the same vaccine serotype from different manufacturers, differences in S1 sequence between different vaccine serials from the same manufacturer, and intra-vaccine differences or quasispecies. Comparing the sequence data of the reisolated viruses with the original vaccine virus, we were able to identify in vivo selection of viral subpopulations as well as mutations. To our knowledge, this is the first report showing selection of a more fit virus subpopulation as well as mutations associated with replication of modified live IBV vaccine viruses in chickens. This information is important for our understanding of how attenuated virus vaccines, including potential vaccines against the SARS-CoV, can ensure long-term survival of the virus and can lead to changes in pathogenesis and emergence of new viral pathogens. This information is also valuable for the development of safer modified live coronavirus vaccines.     

Serotype shift of a 793/B genotype infectious bronchitis coronavirus by natural recombination

An infectious bronchitis coronavirus, designated as ck/CH/LHLJ/140906, was isolated from an infectious bronchitis virus (IBV) strain H120-vaccinated chicken flock, which presented with a suspected infectious bronchitis virus (IBV) infection. A phylogenetic analysis based on the S1 gene clustered ck/CH/LHLJ/140906 with the 793/B group; however, a pairwise comparison showed that the 5′ terminal of the S1 gene (containing hypervariable regions I and II) had high sequence identity with the H120 strain, while the 3′ terminal sequence was very similar to that of IBV 4/91 strain. A SimPlot analysis of the complete genomic sequence, which was confirmed by a phylogenetic analysis and nucleotide similarities using the corresponding gene fragments, suggested that isolate ck/CH/LHLJ/140906 emerged from multiple recombination events between parental IBV strains 4/91 and H120. Although the isolate ck/CH/LHLJ/140906 had slightly higher S1 amino acid sequence identity to strain 4/91 (88.2%) than to strain H120 (86%), the serotype of the virus was more closely related to that of the H120 strain (32% antigenic relatedness) than to the 4/91 strain (15% antigenic relatedness). Whereas, vaccination of specific pathogen-free chickens with the 4/91 vaccine provided better protection against challenge with ck/CH/LHLJ/140906 than did vaccination with the H120 strain according to the result of virus re-isolation. As the spike protein, especially in the hypervariable regions of the S1 domain, of IBVs contains viral neutralizing epitopes, the results of this study showed that recombination of the S1 domain resulted in the emergence of a new serotype.     

Attenuation,safety, and efficacy of a QX-like infectious bronchitis virus serotype vaccine

Avian infectious bronchitis (IB) is a highly contagious disease caused by avian infectious bronchitis virus (IBV), which is a considerable economic threat to the poultry industry. QX-like IBV strains have increasingly emerged in China in recent years. Hence, development of a specific vaccine to guard against their potential threat is important. In this study, we sought to develop an attenuated vaccine strain. First, attenuated QX-like IBV strain SZ130 was created by continuous passage in chicken embryos for 130 generations, and then its safety was tested. We also evaluated the protective efficacy of different doses of SZ130 against challenge with QX-like IBV field strain SD in chickens. SZ130-infected birds did not experience IB-like signs and organ lesions. Additionally, an excellent protective effect of SZ130 vaccination was observed when vaccinated birds were challenged with SD, with no clinical signs or gross lesions, decreased target tissue replication rates, and lower ciliostasis scores in all immunized groups. These findings indicate that attenuated IBV strain SZ130 is highly safe in chicks and may serve as an effective vaccine against the threat posed by QX-like IBV strains.     

Full genome sequence analysis of a newly emerged QX-like infectious bronchitis virus from Sudan reveals distinct spots of recombination

Infectious bronchitis virus (IBV) infection continues to cause economically important diseases in poultry while different geno- and serotypes continue to circulate globally. Two infectious bronchitis viruses (IBV) were isolated from chickens with respiratory disease in Sudan. Sequence analysis of the hypervariable regions of the S1 gene revealed a close relation to the QX-like genotype which has not been detected in Sudan before. Whole genome analysis of IBV/Ck/Sudan/AR251–15/2014 isolate by next generation sequencing revealed a genome size of 27,646 nucleotides harbouring 13 open reading frames: 5′-1a-1b-S-3a-3b-E-M-4b-4c-5a-5b-N-6b-3′. Highest nucleotide sequence identity of 93% for the whole genome was found with the Chinese IBV strain Ck/CH/LHLJ/140906, the Italian IBV isolate ITA/90254/2005 and the 4/91 vaccine strain. Phylogenetic analysis of the S1 gene revealed that the IBV/Ck/Sudan/AR251–15/2014 isolate clustered together with viruses of the GI-19 lineage. Recombination analysis gave evidence for distinct patterns of origin of RNA in the Sudanese isolate in multiple genes. Several sites of recombination were scattered throughout the genome suggesting that the Sudan-QX-like strain emerged as a unique recombinant from multiple recombination events of parental viruses from 4/91, H120 and ITA/90254/2005 genotypes. The Sudanese QX-like isolate is plausibly genetically different from IBV strains previously reported in Africa and elsewhere.     

Origin and characteristics of the recombinant novel avian infectious bronchitis coronavirus isolate ck/CH/LJL/111054

Recombination among infectious bronchitis viruses (IBVs), coupled with point mutations, insertions, and deletions that occur in the genome, is thought to contribute to the emergence of new IBV variants. In this study an IBV, ck/CH/LJL/111054, was isolated from a H120-vaccinated chicken, which presented with a suspected IBV infection. Phylogenetic analysis of the S1 subunit sequence confirmed that strain ck/CH/LJL/111054 is of the Connecticut-type; however, further extensive full-length genomic analysis identified the occurrence of recombination events. Therefore, strain ck/CH/LJL/111054 may have originated from recombination events between Conn- and Mass-like strains at three recombination breakpoints: two located within the nsp3 gene sequence and one in the nsp12 gene sequence. Further, the uptake of the 5′ untranslated regions, nsp2, parts of nsp3, nsp4–11, and parts of nsp 12 from Mass-like virus by ck/CH/LJL/111054 might have resulted in changes in viral replication efficiency rather than antigenic changes, via cross-neutralization analysis with the H120 strain. Recombination events coupled with the accumulation of mutations in the ck/CH/LJL/111054 genome may account for its increased virulence in specific-pathogen free chickens.     

Assembly and immunogenicity of coronavirus-like particles carrying infectious bronchitis virus M and S proteins

Infectious bronchitis virus (IBV) as an avian coronavirus is still posing a persistent and imminent threat to the poultry industry worldwide. Here we report that transfection of Sf9 cells with a single recombinant baculovirus encoding M and S proteins resulted in the assembly of IBV VLPs; this is the first report that S protein plus M protein alone were able to be assembled into VLPs for coronaviruses. We further showed that the generated IBV VLPs could induce humoral immune responses in a level comparable to that of inactivated IBV vaccine, and more importantly the IBV VLPs could elicit significantly higher cellular immune responses than the inactivated IBV vaccine. In summary, the assembly of IBV VLPs with M and S proteins provided a simple strategy for generating VLPs for coronaviruses, and the generated IBV VLPs laid a feasible foundation for the development of an effective vaccine against infection of IBV in the future.     

Development and efficacy of a novel live-attenuated QX-like nephropathogenic infectious bronchitis virus vaccine in China

In this study, we attenuated a Chinese QX-like nephropathogenic infectious bronchitis virus (IBV) strain, YX10, by passaging through fertilized chicken eggs. The 90th passage strain (YX10p90) was selected as the live-attenuated vaccine candidate strain. YX10p90 was found to be safe in 7-day-old specific pathogen free chickens without induction of morbidity or mortality. YX10p90 provided nearly complete protection against QX-like (CH I genotype) strains and partial protection against other two major Chinese genotype strains. YX10p90 also showed no reversion to virulence after five back passages in chickens. An IBV polyvalent vaccine containing YX10p90 was developed and showed that it could provide better protection against major Chinese IBV virulent strains than commercial polyvalent vaccines. In addition, the complete genome sequence of YX10p90 was sequenced. Multiple-sequence alignments identified 38 nucleotide substitutions in the whole genome which resulted in 26 amino acid substitutions and a 110-bp deletion in the 3′ untranslated region. In conclusion, the attenuated YX10p90 strain exhibited a fine balance between attenuation and immunogenicity, and should be considered as a candidate vaccine to prevent infection of Chinese QX-like nephropathogenic IBV.     

Molecular characterization of infectious bronchitis virus in Southwestern China for the protective efficacy evaluation of four live vaccine strains

The high mutation rate of infectious bronchitis virus (IBV) poses a significant threat to the protective efficacy of vaccines. This study aimed at analyzing the S1 genes of IBV field strains isolated in Southwestern China from 2018 to 2020, assessing the pathogenicity of four dominating strains, and evaluating the protective efficacy of four commercial vaccine strains against the endemic representative strains. Thirty-two field strains of IBV were isolated in Southwestern China from 2018 to 2020. Phylogenetic analysis of their S1 genes revealed the nucleotide homology ranged from 64.6% to 100%, and belonged to five genotypes [GI-19 (QX, 53.13%), GI-28 (LDT3-A,15.63%), GI-7 (TW, 12.50%), GI-1 (Mass, 6.23%), GVI-1 (TC07-2, 6.25%)], and two variant groups [variant-3 (3.13%) and variant-5 (3.13%)]. Recombination events between field and vaccine strains or between field strains were identified in the S1 genes of eight IBV field strains. The CK/CH/YNKM/191128 and CK/CH/CQBS/191203 strains of GI-19 showed morbidity rates of 66.7% and 73.7%, respectively, and mortality rates of 13.3% and 33.3%, respectively. Besides, the CK/CH/SCYC/191030 and CK/CH/GZGY/191021 strains of GI-28 caused morbidity rates of 60% and 86.7%, respectively, and mortality rates of 33.3%. The protective efficacy of the four commercial live vaccine strains (4/91, FNO-E55, LDT3-A, and QXL87) ranged from 70% ? 100% and reduced tissue lesions against CK/CH/GZGY/191021 and CK/CH/CQBS/191203 strains. LDT3-A strain was the most effective one but still could not completely prohibit IBV shedding. These findings provide a reference for IBV molecular evolution analysis and control of IB.     

Assembling a global vaccine development pipeline for infectious diseases in the developing world

Commercial realities have drastically reduced private investment in the development of new public health tools, but increased awareness of this situation has resulted in the emergence of a variety of research-based, nonprofit organizations. We reviewed current vaccine developments and developed a framework for efficient research and development investments in this area. We have identified several key "push" and "pull" forces within the vaccine research and product development environment and have examined their impacts on the process. These forces affect the global vaccine pipeline, which is composed of all individual vaccine initiatives and global partnerships (i.e., stakeholders), All of these research and development stakeholders must work together to establish and promote a global, sustainable research and development pipeline that delivers optimal vaccines and immunization technologies.     

Attenuated live infectious bronchitis virus QX vaccine disseminates slowly to target organs distant from the site of inoculation

Infectious bronchitis (IB) is a highly contagious respiratory disease of poultry, caused by the avian coronavirus infectious bronchitis virus (IBV). Currently, one of the most relevant genotypes circulating worldwide is IBV-QX (GI-19), for which vaccines have been developed by passaging virulent QX strains in embryonated chicken eggs. Here we explored the attenuated phenotype of a commercially available QX live vaccine, IB Primo QX, in specific pathogens free broilers. At hatch, birds were inoculated with QX vaccine or its virulent progenitor IBV-D388, and postmortem swabs and tissues were collected each day up to eight days post infection to assess viral replication and morphological changes. In the trachea, viral RNA replication and protein expression were comparable in both groups. Both viruses induced morphologically comparable lesions in the trachea, albeit with a short delay in the vaccinated birds. In contrast, in the kidney, QX vaccine viral RNA was nearly absent, which coincided with the lack of any morphological changes in this organ. This was in contrast to high viral RNA titers and abundant lesions in the kidney after IBV D388 infection. Furthermore, QX vaccine showed reduced ability to reach and replicate in conjunctivae and intestines including cloaca, resulting in significantly lower titers and delayed protein expression, respectively. Nephropathogenic IBVs might reach the kidney also via an ascending route from the cloaca, based on our observation that viral RNA was detected in the cloaca one day before detection in the kidney. In the kidney distal tubular segments, collecting ducts and ureter were positive for viral antigen. Taken together, the attenuated phenotype of QX vaccine seems to rely on slower dissemination and lower replication in target tissues other than the site of inoculation.     

The presence of viral subpopulations in an infectious bronchitis virus vaccine with differing pathogenicity--a preliminary study

There are currently four commercially available vaccines in Australia to protect chickens against infectious bronchitis virus (IBV). Predominantly, IBV causes clinical signs associated with respiratory or kidney disease, which subsequently cause an increase in mortality rate. Three of the current vaccines belong to the same subgroup (subgroup 1), however, the VicS vaccine has been reported to cause an increased vaccinal reaction compared to the other subgroup 1 vaccines. Molecular anomalies detected in VicS suggested the presence of two major subspecies, VicS-v and VicS-del, present in the commercial preparation of VicS. The most notable anomaly is the absence of a 40 bp sequence in the 3'UTR of VicS-del. In this investigation, the two subspecies were isolated and shown to grow independently and to similar titres in embryonated chicken eggs. An in vivo investigation involved 5 groups of 20 chickens each and found that VicS-del grew to a significantly lesser extent in the chicken tissues collected than did VicS-v. The group inoculated with an even ratio of the isolated subspecies scored the most severe clinical signs, with the longest duration. These results indicate the potential for a cooperative, instead of an expected competitive, relationship between VicS-v and VicS-del to infect a host, which is reminiscent of RNA viral quasi-species.     

Complete genome sequences of two avian infectious bronchitis viruses isolated in Egypt: Evidence for genetic drift and genetic recombination in the circulating viruses

Avian infectious bronchitis virus (IBV) is highly prevalent in chicken populations and is responsible for severe economic losses to poultry industry worldwide. In this study, we report the complete genome sequences of two IBV field strains, CU/1/2014 and CU/4/2014, isolated from vaccinated chickens in Egypt in 2014. The genome lengths of the strains CU/1/2014 and CU/4/2014 were 27,615 and 27,637 nucleotides, respectively. Both strains have a common genome organization in the order of 5′-UTR-1a-1b-S-3a-3b-E-M-4b-4c-5a-5b-N-6b-UTR-poly(A) tail-3′. Interestingly, strain CU/1/2014 showed a novel 15-nt deletion in the 4b-4c gene junction region. Phylogenetic analysis of the full S1 genes showed that the strains CU/1/2014 and CU/4/2014 belonged to IBV genotypes GI-1 lineage and GI-23 lineage, respectively. The genome of strain CU/1/2014 is closely related to vaccine strain H120 but showed genome-wide point mutations that lead to 27, 14, 11, 1, 1, 2, 2, and 2 amino acid differences between the two strains in 1a, 1b, S, 3a, M, 4b, 4c, and N proteins, respectively, suggesting that strain CU/1/2014 is probably a revertant of the vaccine strain H120 and evolved by accumulation of point mutations. Recombination analysis of strain CU/4/2014 showed evidence for recombination from at least three different IBV strains, namely, the Italian strain 90254/2005 (QX-like strain), 4/91, and H120. These results indicate the continuing evolution of IBV field strains by genetic drift and by genetic recombination leading to outbreaks in the vaccinated chicken populations in Egypt.