Infectious Bronchitis Virus Vaccine Research Articles

Viral interference between low pathogenic avian influenza H9N2 and avian infectious bronchitis viruses in vitro and in ovo

BackgroundLow pathogenic avian influenza (LPAI) H9N2 and infectious bronchitis virus (IBV) are important pathogens of poultry, causing important economic losses for the sector. Replication interference between these two viruses was described using cell cultures (CC) and embryonated chicken eggs (ECE). Chicken embryo lung (CEL) and ECE were simultaneously or sequentially infected with IBV vaccine strain (H120) and LPAIV-H9N2 (A/Ck/TUN/145/2012) to evaluate viral interactionsin vitro and in ovo, respectively. Real-time RT-PCR was developed to specifically quantify both AIV and IBV genomes as well as viral gene copy numbers during mixed infections. The amount of IL-1 beta, in supernatants of co-infected cell cultures, was determined using an ELISA assay. ResultsQuantitative results of AIV and IBV co-infection showed that interferences between the two viruses yielded decreased viral growth. However, in the case of super-infection, the second virus, either AIV or IBV, induced a decrease in the growth of the first inoculated virus. ConclusionIt appears that either AIV or IBV has a negative impact on the other virus growth when they are inoculated simultaneously or sequentially. The ELISA results showed that higher level of secreted IL-1beta varies, depending on the viral interference conditions between both viruses, during mixed infections.

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.

Preparation and protective efficacy of a chicken embryo kidney cell-attenuation GI-19/QX-like avian infectious bronchitis virus vaccine

Avian infectious bronchitis (IB) is a highly contagious disease, and hazardous to the poultry industry. Immune failure often occurs due to the emergence of new serotypes or field strains antigenically different from the vaccine strains. To prepare a candidate vaccine against the prevalent avian infectious bronchitis virus (IBV) in China, the GI-19/QX-like field isolate Sczy3 was selected as the progenitor strain and attenuated via passaging in chicken embryo kidney (CEK) cells for 100 times. The 100th generation of CEK-adapted strain, designated SczyC100, was safe to use on one-day old specific pathogen-free (SPF) chicken as determined by pathogenicity and virulence reversion test. The efficacies of SczyC100 and two commonly used commercial vaccines (H120 and 4/91) against prevalent GI-19/QX and GI-7/TWI type virulent strains were evaluated. Sczy3C100 effectively reduced the morbidity, mortality, mean lesion scores (MLSs), and viral load of trachea of chickens challenged by GI-19/QX and GI-7/TWI strains. CEK-adapted SczyC100 is therefore a potential vaccine candidate for the control of IB in China.

Field Efficacy of an Attenuated Infectious Bronchitis Variant 2 Virus Vaccine in Commercial Broiler Chickens.

Egyptian poultry suffer from frequent respiratory disease outbreaks associated with Infectious Bronchitis Virus (IBV) variant 2 strains (Egy/VarII). Different vaccination programs using imported vaccines have failed to protect the flocks from field challenge. Recent studies confirmed a successful protection using homologous strains as live attenuated vaccines. In this study, a newly developed live attenuated IB-VAR2 vaccine representing the GI-23 Middle East IBV lineage was evaluated in day-old commercial broilers in an IBV-endemic area. A commercial broiler flock was vaccinated with the IB-VAR2 vaccine at day-old age followed by IB-H120 at day 16. The vaccinated flock was monitored on a weekly basis till the slaughter age. The health status and growth performance were monitored, and selected viral pathogen real-time RT-PCR (rRT-PCR) detection was conducted on a weekly basis. Finally, the flock was compared to a nearby farm with only the classical IB-H120 vaccination program. Results showed that the IB-VAR2 vaccine was tolerable in day-old broiler chicks. The IBV virus rRT-PCR detection was limited to the trachea as compared to its nephropathogenic parent virus. Respiratory disease problems and high mortalities were reported in the IB-H120-only vaccinated flock. An exposure to a wild-type Egy/VarII strain was confirmed in both flocks as indicated by partial IBV S1 gene sequence. Even though the IB-VAR2-vaccinated flock performance was better than the flock that received only IB-H120, the IBV ELISA (enzyme-linked immunosorbent assay) and log2 Haemagglutination inhibition (HI) antibody mean titers remained high (3128 ± 2713 and ≥9 log2, respectively) until the 28th day of age. The current study demonstrates the safety and effectiveness of IB-VAR2 as a live attenuated vaccine in day-old commercial broilers. Also, the combination of IB-VAR2 and classical IBV vaccines confers a broader protective immune response against IBV in endemic areas.

Inactivated infectious bronchitis virus vaccine encapsulated in chitosan nanoparticles induces mucosal immune responses and effective protection against challenge

Avian infectious bronchitis virus (IBV) is one of the most important viral diseases of poultry. The mucosa of upper respiratory tract, specially the trachea, is the primary replication site for this virus. However, conventional inactivate IBV vaccines usually elicit reduced mucosal immune responses and local protection. Thus, an inactivated IBV vaccine containing BR-I genotype strain encapsulated in chitosan nanoparticles (IBV-CS) was produced by ionic gelation method to be administered by oculo-nasal route to chickens. IBV-CS vaccine administered alone resulted in markedly mucosal immune responses, characterized by high levels of anti-IBV IgA isotype antibodies and IFNγ gene expression at 1dpi. The association of live attenuated Massachusetts IBV and IBV-CS vaccine also induced strong mucosal immune responses, though a switch from IgA isotype to IgG was observed, and IFNγ gene expression peak was late (at 5 dpi). Efficacy of IBV-CS was evaluated by tracheal ciliostasis analysis, histopathology examination, and viral load determination in the trachea and kidney. The results indicated that IBV-CS vaccine administered alone or associated with a live attenuated heterologous vaccine induced both humoral and cell-mediated immune responses at the primary site of viral replication, and provided an effective protection against IBV infection at local (trachea) and systemic (kidney) sites.

Resiquimod enhances mucosal and systemic immunity against avian infectious bronchitis virus vaccine in the chicken

Adjuvant enhancing mucosal immune response is preferred in controlling many pathogens at the portal of entry. Earlier, we reported that a toll-like-receptor 7 (TLR7) agonist, resiquimod (R-848), stimulated the systemic immunity when adjuvanted with the inactivated Newcastle disease virus vaccine in the chicken. Here, we report the effect of R-848 when adjuvanted with live or inactivated avian infectious bronchitis virus (IBV) vaccines with special emphasis on mucosal immunity. Specific pathogen free (SPF) chicks (n = 60) were equally divided into six groups at two weeks of age and immunized with either inactivated or live IBV vaccine adjuvanted with or without R-848. Groups that received either PBS or R-848 served as control. A booster was given on 14 days post-immunization (dpi). R-848 enhanced the antigen specific humoral and cellular immune responses when co-administered with the vaccines as evidenced by an increase in the antibody titre in ELISA and stimulation index in lymphocyte transformation test (LTT) till 35 dpi and increased proportion of CD4+ and CD8+ T cells on 21 dpi in the flow cytometry. Interestingly, it potentiated the IgA responses in the tear and intestinal secretions when used with both live and inactivated IBV vaccines. The combination of IBV vaccine with R-848 significantly up-regulated the transforming growth factor beta 4 (TGFβ4) transcripts in the peripheral blood mononuclear cells (PBMCs) than that of the respective vaccine per se. An enhanced secretory IgA response is likely due to the up-regulation of TGFβ4, which is responsible for class switching to IgA. In conclusion, co-administration of R-848 with inactivated or live IBV vaccine enhanced the systemic as well as mucosal immune responses in the chicken.

국내회사와 다국적기업 제조 ELISA 키트의 전염성 기관지염 백신에 따른 항체 검출능 비교

국내회사와 다국적기업 제조 ELISA 키트의 전염성 기관지염 백신에 따른 항체 검출능 비교

Evaluation of golden needle mushroom (<i>Flammulina velutipes</i>) stem waste on pullet performance and immune response

The driving force behind the interest in using natural herbs in poultry production is to eliminate the use of low-dose antibiotics. Therefore, this study was carried out to investigate the effect of Flammulina velutipes mushroom waste (FVW) on performance parameters, relative organ weight, apparent nutrient retention, excreta composition, immune response and serum immunity in pullets. A total of 360 x 10-week-old ISA Brown pullets were randomly assigned to five equal treatment groups, with nine replications of eight birds for each treatment. The dietary treatments included a standard basal diet as control; antibiotic (0.05% flavomycin); 2% FVW; 4% FVW; and 6% FVW. The total experimental duration was 42 days, from 10 weeks to 16 weeks old. Final live weight was higher in FVW groups than in the control and antibiotic groups. No differences were found for average daily feed intake, average daily weight gain and feed conversion ratio during the entire study period. Proventriculus weight and bursa weight were higher in FVW groups. No differences were observed for other inner relative organ weights (liver weight, gizzard weight spleen and abdominal fat weight) compared with the control and antibiotic groups. Dietary inclusion of FVW increased dry matter, crude protein and ether extract retention compared with control and antibiotic groups. Excreta dry matter content was higher and pH lower, in the FVW groups than in the control and antibiotic groups. Antibody titres against Newcastle disease, Infectious bronchitis and Avian influenza virus vaccines were higher in FVW groups. Serum immunoglobulin parameters (IgA, IgG, IgM) were higher in FVW than in the control and antibiotic groups. Flammulina velutipes mushroom waste can be used at inclusion levels up to 6% in pullet rations for better immune response and nutrient retention without hampering normal growth performance. Keywords : Antibody titres, apparent nutrient retention, excreta dry matter, serum immunity

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.

Development and evaluation of novel recombinant adenovirus-based vaccine candidates for infectious bronchitis virus and Mycoplasma gallisepticum in chickens

ABSTRACTAvian infectious bronchitis caused by the infectious bronchitis virus (IBV), and mycoplasmosis caused by Mycoplasma gallisepticum (MG) are two major respiratory diseases in chickens that have resulted in severe economic losses in the poultry industry. We constructed a recombinant adenovirus that simultaneously expresses the S1 spike glycoprotein of IBV and the TM-1 protein of MG (pBH-S1-TM-1-EGFP). For comparison, we constructed two recombinant adenoviruses (pBH-S1-EGFP and pBH-TM-1-EGFP) that express either the S1 spike glycoprotein or the TM-1 protein alone. The protective efficacy of these three vaccine constructs against challenge with IBV and/or MG was evaluated in specific pathogen free chickens. Groups of seven-day-old specific pathogen free chicks were immunized twice, two weeks apart, via the oculonasal route with the pBH-S1-TM-1-EGFP, pBH-S1-EGFP, or pBH-TM-1-EGFP vaccine candidates or the commercial attenuated infectious bronchitis vaccine strain H52 and MG vaccine strain F-36 (positive controls), and challenged with virulent IBV or MG two weeks later. Interestingly, by days 7 and 14 after the booster immunization, pBH-S1-TM-1-EGFP-induced antibody titre was significantly higher (P < 0.01) compared to attenuated commercial IBV vaccine; however, there was no significant difference between the pBH-S1-TM-1-EGFP and attenuated commercial MG vaccine groups (P > 0.05). The clinical signs, the gross, and histopathological lesions scores of the adenovirus vaccine constructs were not significantly different from that of the attenuated commercial IBV or MG vaccines (positive controls) (P > 0.05). These results demonstrate the potential of the bivalent pBH-S1-TM-1-EGFP adenovirus construct as a combination vaccine against IB and mycoplasmosis.

Supplementing dietary rosemary (Rosmarinus officinalis L.) powder and vitamin E in broiler chickens: evaluation of humoral immune response, lymphoid organs, and blood proteins.

The aim of the present study was to evaluate the effect rosemary (Rosmarinus officinalis L.) powder (RP) and vitamin E (VE) at different levels on humoral immunity of broilers during a 42-day production cycle. A total of 270 1-day-old male chicks were assigned to nine groups with three replicates of ten birds each, and diets were supplemented with 0, 0.5, or 1.0% RP and 0, 100, or 200mg/kg VE, respectively. Commercial-inactivated vaccines against avian influenza (AI) and Newcastle disease (ND) viruses, and living infectious bronchitis virus (IBV) vaccine were administered by spray method. Sheep red blood cells (SRBC) were administered subcutaneously. Blood samples were collected from birds 1week after each vaccination to determine antibody titers. At the 42nd day, blood samples were also assessed for globulin level, and lymphoid tissues (thymus, spleen, and bursa) were weighed. Neither antibody titers against viruses nor lymphoid tissues weight were affected by RP and/or VE (P > 0.05) treatments. However, broilers supplemented with 0mg/kg of VE had lower antibody titers against SRBC than those fed 100mg/kg of VE (P < 0.05) at the 24th day. A significant RP × VE interaction effect (P < 0.05) on plasma globulin level was observed. The findings of our study suggest that dietary RP and VE additives can interact and modulate the humoral immunity of broilers, but not sufficiently to improve antibody titers against specific virus during a 42-day production cycle.

Investigation of Cross Neutralization of Egyptian Variant 2 among both Classic and Variant Vaccinal Infectious Bronchitis Viruses

Infectious Bronchitis Virus (IBV) is considered an important virus which may cause major losses in the poultry industry. One of the most important and effective tool that control the infection spread is the vaccination strategy. The present study was undertaken to determine the antigenic relationship between the currently used IBVs, classic and variant vaccinal strains, and the dominating Egyptian variant-2, IBV which could be relatively reflecting the quality of such vaccine. The recommended doses of the monovalent live IBV vaccines of Ma5, H120, 4/91 & CR88, and bivalent vaccine of H120-D274 were administrated via the eye-drop route in groups of 2-week-old SPF-chickens. Three weeks after vaccination, immunized and control chickens were bled, and serum samples were collected. Sera were tested individually for measuring the IBV-Haemagglutination Inhibiting (HI) antibodies against each of IBV-haemagglutinating antigen prepared from the reference classic M41 strain and the Egyptian variant.2 strain (Egy/12/2b spike protein), and IBV- neutralizing antibodies against the Egyptian variant-2 strain. The chicken groups vaccinated with the commercial live vaccines of strains Ma5, H120, 4/91, CR88 and H120-D274 of IBV respectively developed mean serum HI-antibody titers of 6.8, 6.5, 4.9, 5.0 and 5.5 log 2 with the reference classic strain M41-HA antigen, and 3.0, 3.0, 4.4, 4.0, and 4.6 log 2 with the Egyptian variant.2 strain-HA antigen. However, the obtained results revealed utility of VN test more than HI test for assessment of the antigenic relatedness between the vaccinal and challenge strains of IBV as well as testing quality of different commercial live IBV vaccines using the sera of vaccinated chickens against the field strain(s) matched with the homologous strain(s); the antigenic relatedness (r 1 ) between the dominating Egyptian variant-2 strain, and the vaccinal strains, CR88, D274-H120 and H120 of IBV were 33%, 55% and 17% respectively.

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s11262-017-1515-2) contains supplementary material, which is available to authorized users.

Differentiation of infectious bronchitis virus vaccine strains Ma5 and 4/91 by TaqMan real-time PCR

The aim of this study was to develop rapid molecular assays for differentiating vaccine strains Ma5 and 4/91 of the infectious bronchitis virus (IBV). Specific primers and probes for S1 and N genes were designed based on the nucleotide sequences of both vaccine strains. Cross-reactivity was not observed. Assay sensitivity was 2.373 × 103 copies of the Ma5 strain, and 3.852 x 103 copies of the 4/91 strain. Samples belonging to a known genotype demonstrated that the designed assays supported rapid and sensitive detection of Ma5 and 4/91 vaccine strains of IBV.

Astragalus polysaccharides enhance the immune response to avian infectious bronchitis virus vaccination in chickens

Astragalus polysaccharides (APS) are biological macromolecules extracted from Astragalus species that have strong immunoregulatory properties. In this study, APS were employed as an adjuvant for an avian infectious bronchitis virus (IBV) vaccine, and its effects on the cellular immune and humoral immune responses to vaccination in chicken were investigated. One hundred and fifty chicken were randomly divided into five groups (n = 30, each group). The chickens in all groups, except for the unvaccinated control group, were vaccinated with an IBV DNA vaccine. Three of the four vaccinated groups were administered different doses of APS (APSL, 10 mg/kg; APSM, 50 mg/kg; and APSH, 100 mg/kg) after the first vaccination, and the remaining vaccinated group served as a control, without any additional treatment. At 14, 28, and 42 days after the first vaccination, serum anti-IBV antibody titers; peripheral lymphocyte proliferation; and the mRNA expression of IL-1β, IL-2, IL-8, and TNF-α in the spleen were assessed by enzyme-linked immunosorbent assay (ELISA), the cell counting kit-8 (CCK-8), and real time quantitative RT-PCR (qRT-PCR), respectively. At most time points, the titer of IBV-specific antibodies, lymphocyte proliferation, and IL-1β, IL-2, IL-8, and TNF-α mRNA expression levels were higher in three APS groups than in the vaccine control group, and these increases were dose-dependent. These data suggest that APS could be used as an adjuvant for IBV vaccination to provide better protection against IBV infection.

The effect of diatomaceous earth in live, attenuated infectious bronchitis vaccine, immune responses, and protection against challenge

Live virus vaccines are commonly used in poultry production, particularly in broilers. Massive application and generation of a protective local mucosal and humoral immunity with no adverse effects is the main goal for this strategy. Live virus vaccines can be improved by adding adjuvants to boost mucosal innate and adaptive responses. In a previous study we showed that diatomaceous earth (DE) can be used as adjuvant in inactivated vaccines. The aim of this study was to test DE as adjuvant in an Ark-DPI live infectious bronchitis virus (IBV) vaccine after ocular or spray application. Titrating the virus alone or after addition of DE showed that DE had no detrimental effect on the vaccine virus. However, adding DE to the vaccine did not induce higher IgG titers in the serum and IgA titers in tears. It also did not affect the frequency of CD4+ T cells, CD8+ T cells and monocytes/macrophages in the blood and the spleen determined by flow cytometry. In addition, protection generated against IBV homologous challenges, measured by viral load in tears, respiratory signs and histopathology in tracheas, did not vary when DE was present in the vaccine formulation. Finally, we confirmed through our observations that Ark vaccines administered by hatchery spray cabinet elicit weaker immune responses and protection against an IBV homologous challenge compared to the same vaccine delivered via ocular route.

Improved vaccine strategies of infectious bronchitis disease to reduce shedding of virulent virus from infected birds

Infectious bronchitis (IB) threatens the economies of entire nations, by adversely affecting the backyard farmers, poultry producers and poultry industry throughout the world. Infectious bronchitis virus (IBV), a member of the Coronaviridae family, is probably one of the most avian pathogens prevalent in the poultry industry worldwide. In the field which presents difference structures, especially in the S1 spike protein. Complicated immune response against IBV due to few similarities between serotypes. High mutation rate of the virus, in addition to the management and environmental factors, compromise the efficacy of the available vaccines and difficult the control of disease. Prepared effective IB vaccines from any IBV strain stimulates the immunity widely against morbidity and mortality after challenge with virulent IBV strains. On the other hand, vaccinates with live IBV vaccines do not reduce infection or viral shedding after challenge. In order to compare the prepared IBV vaccines taken four different IBV genotypes to determine stimulating immunity and the amount of viral shedding after challenge, two hundred fifty one day broiler chicks divided into five groups and vaccinated with oil-adjuvant vaccines prepared of Iraqi isolates (II, I-II, I- III and I-IV). different inactivated IB viruses including strains QX, CH Baghdad M 2014, CH Baghdad F1 2013, CH Babylon F2 2013, and an allantoic fluid control. Using a hemagglutination inhibition test for serum analysis after challenge with virulent IBV (Variant2) to knowledge antibody content against each of the vaccine antigens, Monitored the vaccinated birds daily for of morbidity and mortality were recorded, and used PCR to determine the amount of viral copy of examined clinical samples in selected periods of viral shedding. All vaccines used in the study gave good protection against morbidity and mortality, except the control group. Homologous vaccines gave good protection and lowest viral shedding in the trachea, kidney and feces compared to the heterogeneous vaccines.Key word: infectious bronchitis disease, inactivated vaccines, hemagglutination inhibition, viral load, challenge.

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.

Molecular characterization and phylogenetic analyses of virulent infectious bronchitis viruses isolated from chickens in Eastern Saudi Arabia.

Infectious bronchitis virus (IBV) is one of the major respiratory viral threats for chickens. Despite the intensive application of IBV vaccines, several outbreaks have been reported worldwide. Here, we report several IBV outbreaks in thirteen poultry farms in Eastern Saudi Arabia (ESA) from 2013 to 2014. The main goals of the current study were as follows: (1) isolation and molecular characterization of the currently circulating strains in ESA (Al-Hasa, Dammam, and Buqayq) and (2) evaluation of the immune status of these birds to IBV. To achieve our goals, tissue specimens (trachea, lungs, liver, kidney and cecal tonsils) and sera were collected. High morbidity up to 100% and mortality ranging from 18 to 90% were reported. Severe infection was observed in the trachea, bronchi, and kidneys of the infected birds. IBV strains were isolated using embryonated chicken eggs. The isolated viruses induced hemorrhage, dwarfing and death of the inoculated embryos 3–5 days post-infection. The circulating IBV strains were identified by sequencing the partial IBV-N and IBV-S1 genes. These viruses showed 95% sequence identity to Indian, Italian, Egyptian and Chinese strains and were quite distinct from the locally used vaccines on the genomic level. Interestingly, high antibody titers against IBV were reported in some of these farms, suggesting the presence of new virulent strains in ESA. The seroconversion of infected birds was reported among the affected flocks. In conclusion, very virulent IBV strains are currently circulating in ESA. Further studies are currently in progress to molecularly characterize these IBV strains. Electronic supplementary materialThe online version of this article (doi:10.1007/s13337-017-0375-7) contains supplementary material, which is available to authorized users.

Different evolutionary trajectories of vaccine-controlled and non-controlled avian infectious bronchitis viruses in commercial poultry.

To determine the genetic and epidemiological relationship of infectious bronchitis virus (IBV) isolates from commercial poultry to attenuated live IBV vaccines we conducted a phylogenetic network analysis on the full-length S1 sequence for Arkansas (Ark), Massachusetts (Mass) and Delmarva/1639 (DMV/1639) type viruses isolated in 2015 from clinical cases by 3 different diagnostic laboratories. Phylogenetic network analysis of Ark isolates showed two predominant groups linked by 2 mutations, consistent with subpopulations found in commercial vaccines for this IBV type. In addition, a number of satellite groups surrounding the two predominant populations were observed for the Ark type virus, which is likely due to mutations associated with the nature of this vaccine to persist in flocks. The phylogenetic network analysis of Mass-type viruses shows two groupings corresponding to different manufacturers vaccine sequences. No satellite groups were observed for Mass-type viruses, which is consistent with no persistence of this vaccine type in the field. At the time of collection, no vaccine was being used for the DMV/1639 type viruses and phylogenetic network analysis showed a dispersed network suggesting no clear change in genetic distribution. Selection pressure analysis showed that the DMV/1639 and Mass-type strains were evolving under negative selection, whereas the Ark type viruses had evolved under positive selection. This data supports the hypothesis that live attenuated vaccine usage does play a role in the genetic profile of similar IB viruses in the field and phylogenetic network analysis can be used to identify vaccine and vaccine origin isolates, which is important for our understanding of the role live vaccines play in the evolutionary trajectory of those viruses.