Genotypic and phenotypic characterization of the California 99 (Cal99) variant of infectious bronchitis virus

The California 99 (Cal99) variant of infectious bronchitis virus (IBV) was first recovered in 1999 from vaccinated broiler chicken flocks in Central California. The S1 hypervariable region of Cal99 genome was most closely related to Arkansas (Ark) serotype viruses. In this study, the complete genome of Cal99 was sequenced, and the structural protein genes were compared with those of commonly used IBV vaccines as well as those of isolates from naturally occurring outbreaks in different parts of the world, to elucidate potential sources of genetic material. Based on sequence comparison, the prototype Cal99 virus is similar to the apathogenic ArkDPI virus, except in the S1 gene and stretches of sequence in the S2 and M structural protein genes, which are more related to Connecticut (Conn) and Massachusetts (Mass) strain viruses, respectively. We speculate that these two fragments came from a Conn and a Mass virus, respectively, and were incorporated into a virus largely derived from ArkDPI. Since Ark, Conn and Mass strains have been simultaneously used as live vaccines in California, both point mutations and recombination among vaccine strains may have contributed to the emergence of the Cal99 variant virus. Analysis of the structural protein genes of six Cal99 isolates demonstrated that viruses of this serotype may differ substantially in the non-S1 structural genes. Finally, we performed a challenge study with Cal99 and demonstrated that the virus causes late-onset respiratory disease, with a severity comparable to that of the M41 IBV challenge strain.     

Phylogenetic analysis of the S1 glycoprotein gene of infectious bronchitis viruses isolated in China during 2009–2010

As part of our ongoing surveillance program, 40 field strains of avian infectious bronchitis virus (IBV) were isolated from dead or diseased chicken flocks in different areas of China between 2009 and 2010. S1 glycoprotein genes of these strains were sequenced and analyzed with 38 strains published in GenBank. S1 genes of these isolated strains and the vaccine strains showed nucleotide homologies ranging from 65.2 to 82% and amino acid homologies ranging from 58.4 to 81.9%. Meanwhile, Chinese IBV strains isolated in this study, which were mainly nephropathogenic, could be separated into six variant lineages (CH I–CH VI), and current vaccine strains used in China formed Mass variant lineage that is evolutionarily distant from Chinese isolates. Moreover, CK/CH/GD/NC10, CK/CH/GD/KP10, and our previous isolates TC07-2 formed the CH VI lineage, showing larger evolutionary distances from other strains. Taken together, these findings suggested that various variant lineages were co-circulating in China now, and appeared to be continuously evolving, alternative indigenous vaccines indeed need for effective control of IB in China.     

Sequence analysis of infectious bronchitis virus isolates from the 1960s in the United States

To better understand the molecular epidemiology of infectious bronchitis virus (IBV) in the United States following the introduction of commercial IBV vaccines, we sequenced the S1 and N structural protein genes of thirteen IBV field isolates collected in the 1960s. Analysis of the S1 sequence showed that seven isolates were of the Massachusetts (Mass) genotype, five were SE17, and one was of the Connecticut (Conn) genotype, suggesting that these three IBVs were circulating in commercial poultry raised in different regions in the United States during the 1960s. The S1 genes of Mass-type isolates had high levels of sequence variation, representing 81.3-81.9 % nucleotide (nt) and 77.3-78.7 % amino acid (aa) identity when compared to those of the SE17-type isolates. In contrast, the N genes from the same isolates were less variable (>92 % nt and >93 % aa identity) when compared to those of the SE17-type isolates. Phylogenetic analysis based on the S1 gene indicated that one isolate (L748) was more closely related to the Mass type. In contrast, phylogenetic analysis based on the N gene showed that L748 was more closely related to the SE17 type, indicating that there had been exchange of S1 genetic materials between Mass- and SE17-like viruses. In addition, the Mass-type isolates had high levels of sequence identity in the S1 gene compared with widely used modified live vaccines (Mass41, Ma5 and H120) and modern field strains from the USA and other countries, suggesting a common ancestor.     

Protection of chickens vaccinated with combinations of commercial live infectious bronchitis vaccines containing Massachusetts,Dutch and QX-like serotypes against challenge with virulent infectious bronchitis viruses 793B and IS/1494/06 Israel variant 2

Infectious bronchitis virus (IBV) is a coronavirus which affects chickens of all ages. IBV mainly causes respiratory disease but can also result in reduced weight gain, reduced egg production, increased frequency of abnormal eggs and increased rates of mortality. Vaccination is the most important way to control the disease. Nevertheless, novel strains of infectious bronchitis (IB) continue to emerge in the field. In order to respond promptly, combinations of existing IB vaccines are frequently tested to see whether they can provide cross-protection. The efficacy of a combination of vaccines based on Massachusetts, Dutch and QX-like IB strains against emerging IB Israel variant 2 and IB 793B strains was assessed by means of four challenge studies. At least 80% of the birds vaccinated with IB H120 (Mass type) combined with IB D274 (Dutch type) followed by a QX-like IB vaccine booster or vaccinated with a combination of IB H120, IB D274 and QX-like IB were protected against a challenge with IB 793B. In addition, IB 1263 (Mass type) boosted by QX-like IB showed an 85% protection following challenge with IB 793B. A combination of IB H120 and IB D274 boosted by QX-like IB vaccine conferred 70% protection whilst H120 and IB D274 combination on its own showed 61.1% protection against Israel variant 2 challenge. IB 1263 boosted by a QX-like IB vaccine showed 50% protection against IB Israel variant 2. Therefore, it can be concluded that a combination of the IB H120, IB D274 and QX-like IB confers broad protection against different non-related virulent IB strains.     

A novel variant of avian infectious bronchitis virus resulting from recombination among three different strains

Summary An antigenic variant of avian infectious bronchitis virus (IBV), a coronavirus, was isolated and characterized. This strain, CU-T2, possesses a number of unusual features, which have not been previously observed in IBV. The S1 glycoprotein of CU-T2 carries virus-neutralizing and serotype-specific epitopes of two IBV serotypes, Arkansas (Ark) and Massachusetts (Mass). Sequence analysis revealed that the virus, originally an Ark serotype, has acquired the Mass-specific epitope by mutation(s). This provides evidence that point mutations may lead to generation of IBV antigenic variants in the field. It was further observed that two independent recombination events involving three different IBV strains had occurred in the S2 glycoprotein gene and N protein gene of CU-T2, indicating that genomic RNA recombination in IBV may occur in multiple genes in nature. It was especially significant that a sequence of Holland 52 (a vaccine strain) had replaced half of the N gene of CU-T2. This proves that recombination among vaccine strains is contributing to the generation of IBV variants in the field. Based on these observations it is predicted that every IBV field isolate could have unique genetic nature. Therefore, several recently reported diagnostic and serotyping methods of IBV which are based on dot-blot hybridization, restriction fragment length polymorphism (RFLP), and polymerase chain reaction (PCR), may not reveal the true antigenic and/or genetic nature of IBV isolates, and may in fact yield misleading information.     

Molecular Characterization of Three New Avian Infectious Bronchitis Virus (IBV) Strains Isolated in Quebec

Three unrecognized field isolates of Infectious Bronchitis Virus (IBV) were recovered from commercial broiler chickens vaccinated with live Mass viral strain (H120). These isolates were identified by immunofluorescence using monoclonal antibodies produced against reference serotypes: Mass, Conn, and Ark. RT-PCRs were performed on viral RNAs to amplify S1 gene using a specific set of primers S1OLIGO3 and S1OLIGO5. Restriction polymorphism (RFLP) of PCR products was determined by the use of HaeIII restriction enzyme. As expected, patterns of PCR products were different from common pattern of strains assigned to Mass serotype M41, Beaudette, H120, and Florida. Molecular analysis showed a nucleotide insertion in hypervariable region one (HVR-1) of S1 gene of only Quebec isolates (Qu16, Qu_mv and Q_37zm). However, New Brunswick IBV isolate (NB_cp) did not display these insertions. Major amino acid changes involved insertion of two stretches (aa_118–119: Arg–Ser and aa_141–145: Sys–Ser–Asn–Ala–Ser–Cys) located at N-terminal and C-terminal regions of HVR-2. It is speculated that cysteine residue located upstream and downstream of Cys–Ser–Asn–Ala–Ser–Cys segment might be involved in the formation of loop structure and disulfide bond that could trigger important epitope changes. Insertion of new NXT and NXS (XP) glycosylation motifs scattered along S1 region and insertion of cysteine residues in HVR are contributing to the antigenic shifting of Quebec isolates. Fragment insertions were thought to be induced by inter-serotype recombination between vaccine strain (H120) that belongs to Mass serotype and another strain belonging to Ark serotype. Phylogenetic tree based on amino acid sequences showed that Quebec isolates formed a new phylogenetic cluster.     

Variability in biological behaviour,pathogenicity, protectotype and induction of virus neutralizing antibodies by different vaccination programmes to infectious bronchitis virus genotype Q1 strains from Chile

In the period from July 2008 to 2010, a disease episode resulting in serious economic losses in the major production area of the Chilean poultry industry was reported. These losses were associated with respiratory problems, increase of condemnations, drops in egg production and nephritis in breeders, laying hens and broilers due to infections with infectious bronchitis virus (IBV). Twenty-five IBV isolates were genotyped and four strains were selected for further testing by pathotyping and protectotyping. Twenty-four IBV isolates were of the Q1 genotype. The experiments also included comparing the ability of six vaccination programmes to induce virus neutralizing antibodies (VNA) in layers against four selected Chilean strains. Despite the high genetic homology in the S1 gene between the four strains, the heterogeneity in biological behaviour of these different Q1 strains was substantial. These differences were seen in embryonated eggs, in cell culture, in pathogenicity and in level of cross-protection by IBV Massachusetts (Mass) vaccination. This variability underlines the importance of testing more than one strain per serotype or genotype to determine the characteristics of a certain serotype of genotype. The combination of Mass and 793B vaccine provided a high level of protection to the respiratory tract and the kidney for each strain tested in the young birds. The combination of broad live priming using Mass and 793B vaccines and boosting with multiple inactivated IBV antigens induced the highest level of VNA against Q1 strains, which might be indicative for higher levels of protection against Q1 challenge in laying birds.     

Protection following simultaneous vaccination with three or four different attenuated live vaccine types against infectious bronchitis virus

ABSTRACT

Two or more different live attenuated infectious bronchitis virus (IBV) vaccine types are often given to broilers to induce homologous protection as well as to broaden protection against other IBV types in the field. However, the ability of broilers to respond to three or four different antigenic types of IBV vaccine has not been examined experimentally. In this study, we vaccinated one-day-old broiler chicks by eyedrop with three or four different IBV vaccine types simultaneously. The presence and relative amount of each vaccine was examined in all of the birds by IBV type-specific real-time RT–PCR at 5 days post-vaccination and each vaccine was detected in all of the birds given that vaccine. The birds were challenged at 28 days of age and protection was measured by clinical signs, virus detection and by ciliostasis. Birds vaccinated with three different IBV types (Ark, Mass and GA98) were protected against challenge with each of those IBV types and were partially protected against challenge with the GA08 virus. Birds vaccinated with four different IBV types (Ark, Mass, GA98 and GA08) were protected against challenge with each of those IBV types with the exception of Mass challenged birds which clearly had 3/11 birds not protected based on individual ciliostasis scores, but had an average ciliostasis score of >50% which is considered protected. The results are important for the control of IBV because they indicate that simultaneous vaccination with up to four different IBV vaccine types can provide adequate protection against challenge for each type.     

Sequence Comparison of Avian Infectious Bronchitis Virus S1 Glycoproteins of the Florida Serotype and Five Variant Isolates from Georgia and California

The infectious bronchitis virus (IBV) spike glycoprotein S1 subunit is required to initiate infection and contains virus-neutralizing and serotype-specific epitope(s). Reported are the S1 gene nucleotide and predicted amino acid sequences for the Florida 18288 strain and isolates GA-92, CV-56b, CV-9437, CV-1686, and 1013. These sequences were compared with previously published gene sequences of IBV strains, and phylogenetic relationships are reported. The S1 amino acid sequence of Florida 18288 was 94.9% similar to the Connecticut strain, and GA-92 was 92.8% similar to the Arkansas 99 strain. S1 amino acid sequences of the California variants, CV-56b, CV-9437, and CV-1686, were 97.6–99.3% similar to one another and only 76.6%–76.8% similar to the Arkansas-type strains. Isolate 1013, also from California, was 84.0% similar to Ark DPI and 77.9% similar to CV-56b. When comparing 19 viruses isolated from the United States, sequence variations were observed between amino acids 55–96, 115–149, 255–309, and 378–395. Similar regions are reported to be involved in virus-neutralizing and/or serotype-specific epitopes. These data demonstrate that variant IBV strains continue to emerge, and unique variants may circulate among poultry in geographically isolated areas. This revised version was published online in July 2006 with corrections to the Cover Date.     

Induction of IBV strain-specific neutralizing antibodies and broad spectrum protection in layer pullets primed with IBV Massachusetts (Mass) and 793B vaccines prior to injection of inactivated vaccine containing Mass antigen

In an initial study in specified pathogen free (SPF) chickens, a heterologous virus neutralizing (VN) antibody response to IBV variants Q1, Variant 2 (Var 2), D388/QX (D388), D274 and Arkansas (DPI) was observed using a vaccination programme incorporating two different live-attenuated IBV vaccines, followed by boosting with an inactivated vaccine containing IBV Massachusetts (Mass) antigen. Therefore, a more detailed study was undertaken in SPF layer-type chickens primed with IBV Mass and 793B vaccines. The efficacy of single or repeated vaccination with a multivalent inactivated vaccine containing IBV antigen was determined against challenge with five virulent IBVs: Mass (M41), 793B (4/91), D388, Q1 and Var 2. The parameters assessed were serological response, respiratory signs, egg production, post mortem abnormalities in the reproductive organs and abdomen, and incidence of IBV antigen in kidneys. Increased VN titres were recorded against the five IBV challenge strains, with a significantly higher level of protection against drops in egg production following challenge. The difference between one or two vaccinations with inactivated vaccine was not significant in terms of egg production. However, a significantly increased level of protection was seen in the lower percentage of hens with free yolk in the abdomen and/or peritonitis post challenge with IBV variants, D388, Q1 and Var 2 not included in the vaccination programme. A lower incidence of acute, degenerated ovaries was found in groups given one injection of inactivated vaccine following live priming, and this was significantly lower than in groups given only live priming.     

Molecular cloning and sequence comparison of the S1 glycoprotein of the Gray and JMK strains of avian infectious bronchitis virus

The nucleotide sequences of S1 glycoprotein genes of the Gray and JMK strains of avian infectious bronchitis virus (IBV) were determined and compared with published sequences for IBV. The IBV Gray and JMK strains had 99% nucleotide sequence similarity. The overall nucleotide sequence similarity of the Gray and JMK strains compared with other IBV strains was between 82.0% and 87.4%. The similarity of the predicted amino acid sequence for the S1 glycoproteins of the Gray and JMK strains was 98.8%. Six of the 10 differences in the amino acid sequence were found between residues 99 and 127, suggesting a possible role for that region in the tissue trophisms of the viruses. The S1 glycoprotein of the Gray and JMK strains had 79.5%–84.6% amino acid similarity with the published sequence of other IBV strains. Serine instead of phenylalanine was observed in the protease cleavage site between the S1 and S2 glycoprotein subunits for the Gray and JMK strains, which was similar to the published sequence for the Ark99 and SE17 strains. The significance of that amino acid change is not known. Based on the nucleotide sequence of the Gray and JMK strains, theBsmAI restriction enzyme was selected by computer analysis and was used in restriction fragment length polymorphism analysis to differentiate the two strains.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers GRAYS1=L14069 and JMKS1=L14070.     

Combined infectious bronchitis virus Arkansas and Massachusetts serotype vaccination suppresses replication of Arkansas vaccine virus

Polyvalent infectious bronchitis virus vaccination is common worldwide. The possibility of vaccine interference after simultaneous combined vaccination with Arkansas (Ark) and Massachusetts (Mass)-type vaccines was evaluated in an effort to explain the high prevalence of Ark-type infectious bronchitis virus in vaccinated chickens. Chickens ocularly vaccinated with combinations of Ark and Mass showed predominance of Mass vaccine virus before 9 days post-vaccination (DPV) in tears. Even when Mass and Ark vaccines were inoculated into separate eyes, Mass vaccine virus was able to outcompete Ark vaccine virus. Although Mass vaccine virus apparently had a replication advantage over Ark vaccine in ocular tissues, Ark vaccine virus appeared to have an advantage in spreading to and/or replicating in the trachea. When chickens vaccinated with Ark or Mass vaccine were housed together, Mass vaccine virus was able to spread to Ark-vaccinated chickens, but the Ark vaccine was not detected in Mass-vaccinated chickens. Only Mass vaccine was detected in tears of sentinel birds introduced into groups receiving both vaccines. Furthermore, Ark vaccine virus RNA was not detectable until 10 DPV in most tear samples from chickens vaccinated with both Ark and Mass vaccines at varying Ark vaccine doses, while high concentrations of Mass virus RNA were detectable at 3–7 DPV. In contrast, Ark vaccine virus replicated effectively early after vaccination in chickens vaccinated with Ark vaccine alone. The different replication dynamics of Ark and Mass viruses in chickens vaccinated with combined vaccines did not result in reduced protection against Ark challenge at 21 DPV. Further studies are needed to clarify if the viral interference detected determines differences in protection against challenge at other time points after vaccination.     

Analysis of S1 gene of avian infectious bronchitis virus isolated in southern China during 2011–2012

Sixty-two strains of avian infectious bronchitis virus (IBV) were isolated from diseased chickens at different farms in southern China during 2011–2012, and 66.1 % of the isolated strains were associated with typical nephritis. Analysis of the S1 gene sequences amplified from the 62 isolated strains together with 40 reference strains published in Genbank showed nucleotide homologies ranging from 63.5 to 99.9 % and amino acid homologies ranging from 57.9 to 100 %. Phylogenetic analysis revealed that all Chinese IBV strains were clustered into six distinct genetic groups (I–VI). Most of the isolated strains belonged to group I, and the isolation of group V strains was increased compared with an earlier period of surveillance. Current vaccine strains used in China (H120, H52, W93, and Ma5) formed the group Mass which is evolutionarily distant from Chinese isolates. Alignment of S1 amino acid sequences revealed polymorphic and diverse substitutions, insertions, and deletions, and the S1 protein of major pandemic strains contained 540 amino acids with a cleavage site sequence of HRRRR or RRF(L/S)RR. Further analysis showed that recombination events formed a new subgroup. Taken together, these findings suggest that various IBV variants were co-circulating and undergoing genetic evolution in southern China during the observation period. Therefore, long-term continuing surveillance is significantly important for prevention and control of IBV infection.     

Genetic diversity of avian infectious bronchitis coronavirus strains isolated in China between 1995 and 2004

Summary. Twenty-six avian infectious bronchitis (IB) viruses (IBV) were isolated from outbreaks in chickens in China between 1995 and 2004. They were characterized by comparison with twenty-six Chinese reference strains and five other IBV strains. Chinese IBVs, which were mainly nephropathogenic, were placed into seven genotypes. Fourteen Chinese IBV isolates were placed in genotype I, having small evolutionary distances from each other. Genotype II included 6 strains that were isolated in the 1990s in China. Genotype III consisted of eight Chinese isolates that showed close relationship with Korean IBV isolates. Another eight IBV isolates clustered in genotype IV and showed larger evolutionary distances. The Massachusetts serotype was present in China in 1990s and was in a separate genotype. Two isolates, HN99 and CK/CH/LHN/00I, which might be a reisolation of vaccine strains, clustered into genotype VI. Four Chinese IBV isolates formed another genotype and showed larger evolutionary distances from other Chinese IBV genotypes (genotype VII). IBVs in same genotypes showed more than 90% amino acid sequence similarities, whereas most of the viruses in different genotypes showed less than 90%. The results showed that IBVs in China came from genetic changes both in IBV populations that existed before the advent of vaccination and in the viruses that were introduced through live vaccines. IBVs showing various genetic differences are cocirculating in China.     

Evaluation of the genetic variability found in Brazilian commercial vaccines for infectious bronchitis virus

Infectious bronchitis virus (IBV) is currently one of the most important pathogens in the poultry industry. The H120 and Ma5 are the only viral strains approved by the Brazilian government as the constituent of vaccines. Despite the systematic vaccination in Brazil, IBV has not yet been controlled and diseases associated with this virus have been reported in vaccinated chickens. Here, we investigated the genetic variability of H120 and Ma5 strains present in the IBV vaccines from different Brazilian manufacturers. We performed DNA sequencing analyses of the S1 spike glycoprotein gene to investigate its genetic variability and the presence of viral subpopulations among vaccines, between batches, and also in each vaccine after a single passage was performed in chicken embryonated eggs. Our results revealed up to 13 amino acid substitutions among vaccines and some of them were localized in regions of the S1 glycoprotein that play a role in virus–host interaction. Secondary nucleotide peaks identified in the chromatogram for the S1 gene sequence revealed that all original vaccines (H120 and Ma5) were composed by different subpopulations of IBV. Moreover, new viral subpopulations were also found in vaccines after a single passage in chicken embryonated eggs. These findings indicate that H120 and Ma5 viral strains used in vaccines market in Brazil can still mutate very rapidly during replication, leading to amino acid substitutions in proteins involved in the stimulation of the immune response, such as the S1 glycoprotein. Therefore, our data suggest that the genetic variability of these viral strains should be taken into consideration to ensure an effective immune response against IBV.