CONTROL OF INFECTIOUS BRONCHITIS IN POULTRY

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0221845
• Project Start Date: Oct 1, 2010
• Project End Date: Sep 30, 2015
• Program Code: [(N/A)]- (N/A)

Recipient Organization
AUBURN UNIVERSITY
108 M. WHITE SMITH HALL
AUBURN,AL 36849

Performing Department
College of Veterinary Medicine

Non Technical Summary
Infectious bronchitis virus (IBV)continues to be a common and economically important contributor to overall disease losses in poultry in the U.S. Economic losses due to IBV include reduced feed conversion, mortality, and increased condemnations at processing from secondary bacterial infections, as well as declines in egg production. Most IBV isolates (≥60%) obtained from outbreaks of respiratory disease in south eastern, mid-Atlantic, and south central regions of the U.S. correspond to Ark-type IBV despite extensive vaccination with serotype-specific (ArkDPI-derived) vaccines. Our experimental data have demonstrated high genetic and phenotypic heterogeneity within the IBV virus populations of all available ArkDPI-derived live attenuated vaccines. We have also shown that the vaccine's predominant virus phenotype is rapidly negatively selected during a single passage in chickens. Finally, we have demonstrated that IBV Ark vaccine strains undergo intraspatial variation during the invasion process; i.e. the predominant genotype/phenotype further changes during host invasion as the microenvironment of distinct tissues exerts selective pressure on the replicating virus population. Intraspatial variation of IBV vaccine strains also results in the host immune response having to "chase" or adapt to a continuously changing target (i.e. new predominant phenotypes). Consequently, the immune response is less efficient providing an explanation to the increased persistence of ArkDPI vaccine viruses observed in commercial chickens. We have identified Ark-IBV populations showing distinct non-synonymous changes in the S1 gene (encoding the IBV's attachment protein) that become predominant in the environments of the chicken upper respiratory tract as well as other tissues shortly after exposure. This novel knowledge allows us to propose the development of "host customized" recombinant vaccines; i.e. encoding the S1 protein of the predominant IBV population that is selected by the host and actually presented to the immune system. We will develop recombinant vaccines vectoring codon optimized S1 transgenes synthesized based on the sequences of the predominant virus population replicating in distinct tissues of chickens inoculated with Ark-DPI strains. As expression model we propose to use the replication defective recombinant adenovirus (Ad) technology previously developed in our laboratories. Immunogenicity and protection conferred by vaccine constructs expressing distinct IBV S1 proteins will be evaluated. The improvement of Ark-type vaccines is essential for an effective control of IBV in the poultry industry. A recombinant vaccine will overcome the problems inherent to attenuated ArkDPI-derived vaccines. The novel concept of host-customized vaccines will conceivably have an impact in general vaccinology.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3220	1090	10%
311	3220	1101	40%
311	4030	1090	40%
311	4030	1101	10%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3220 - Meat-type chicken, live animal; 4030 - Viruses;

Field Of Science
1090 - Immunology; 1101 - Virology;

Keywords

infectious bronchitis virus,

vaccine

Goals / Objectives
CRIS retrieval A CRIS retrieval on the terms of "infectious bronchitis" and "vaccine" was conducted on March 23, 2010. This search returned 40 projects. One of them (originating in our own laboratory) pertained to the specific goal 1 of this project but did not provide overlap or duplication of the proposed research. The proposed research proposal is based on our recent findings on IBV intraspatial variation. This phenomenon was reported for the first time by our group. Thus, the proposed research will be new and not redundant with respect to related projects. Goals The overall aim of this project addresses Alabama's poultry production system. Poultry "is the state's most important agricultural commodity," with a ~$9 billion annual economic impact. Respiratory disease continues to be the most common and economically important contributor to overall disease losses in poultry in the U.S. One measure of the impact of respiratory disease is the slaughter condemnation losses reported as due to airsacculitis and septicemia. For example, during the week ending 6 December 2009, post mortem condemnations of broilers due to airsacculitis and septicemia in Alabama represented 89.4% of all Alabama broiler condemnations (USDA National Agricultural Statistics Service). Infectious bronchitis virus is endemic in all major poultry producing areas in the world. Economic losses due to IBV include reduced feed conversion, mortality, and condemnations at processing from secondary bacterial infections, as well as declines in egg production. Most IBV isolates (≥60%) obtained from outbreaks of respiratory disease in south eastern, mid-Atlantic, and south central regions of the U.S. correspond to Ark-type IBV despite extensive vaccination with serotype-specific (ArkDPI-derived) vaccines. In this prject we propose to (1) evaluate pathogenicity and immunogenicity of Ark-type IBV subpopulations; i.e. predominant geno/phenotypes within Ark IBV strains; and (2) to develop a recombinant vaccine vectoring codon optimized S1 transgenes synthesized based on the sequences of predominant virus geno/phenotypes to protect chickens against IBV.

Project Methods
1) We will further characterize IBV phenotypes that become predominant in different tissues of the host. Characterization of predominant phenotypes will be based on sequence analysis of the S1 gene. Pathogenicity will be evaluated by clinical signs, viral shedding, pathological and histopathological changes. Immunogenicity will be evaluated by determining mucosal and systemic immune responses (6). 2) We will evaluate recombinant constructs vectoring codon optimized S1 transgenes synthesized based on the sequences of the predominant virus population replicating in distinct tissues of chickens inoculated with Ark-DPI strains. As expression model we propose to use the replication competent adenovirus (RCA)-free recombinant adenovirus (Ad) technology, previously developed in our laboratories for use in chickens (3-5). Immunogenicity and resistance conferred by vaccine constructs expressing distinct S1 proteins will be evaluated using the in ovo and mucosal (ocular, spray) delivery routes. Antibody responses and cross-protection [different S1 proteins versus homologous and heterologous strains (including Ark wild and vaccine)] will initially be assessed in serum neutralization assays. In subsequent animal trials protection against challenge will be evaluated by clinical signs, viral shedding, pathological and histopathological changes, as well as mucosal and systemic immune responses. The improvement of Ark-type vaccines is essential for an effective control of IBV in the poultry industry. A recombinant vaccine expressing an immunodominant epitope will overcome the problems inherent to attenuated ArkDPI-derived vaccines. To accomplish these goals we will further collaborate with investigators at Vaxin, a vaccine development company located in Birmingham Al.

Progress 10/01/10 to 09/30/15

Outputs
Target Audience:Poultry veterinarians, poultry industry, pharmaceutical industry associated with poultry Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?2 PhD students were involved How have the results been disseminated to communities of interest?Results have been disseminated to scientists and poultry industry during multiple meetings and published articles What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. We developed a replication defective adenovirus recombinant vaccine vectoring the S1 gene of IBV 2. We developed a Newcastle disease virus recombinant vaccine vectoring the S2 gene of IBV 3. We developed an improved attenuated Ark-type vaccine

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Ghetas, A.M., G.E. Thaxton, C. Breedlove, V.L. van Santen, H. Toro (2015). Effects of Adaptation of Infectious Bronchitis Virus Arkansas Attenuated Vaccine to Embryonic Kidney Cells. Avian Diseases 59:106-113.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Eunice N. Ndegwa, Samantha N. Bartlett, Haroldo Toro, Kellye S. Joiner & Vicky L. van Santen (2015). Combined infectious bronchitis virus Arkansas and Massachusetts serotype vaccination suppresses replication of Arkansas vaccine virus. Avian Pathology 44:408-420
• Type: Journal Articles Status: Published Year Published: 2015 Citation: H. Toro, V. L. van Santen, A. M. Ghetas, and K. S. Joiner (2015) Cross-Protection by Infectious Bronchitis Viruses Under Controlled Experimental Conditions. Avian Diseases 59:532-536

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? We will evaluate protection conferred by kidney cell adapted Ark IBV. We will also continue working on a recombinant vaccine to protect chicken populations against infectious bronchitis.

Impacts
What was accomplished under these goals? The population structure of an embryo-attenuated infectious bronchitis virus (IBV) Arkansas (Ark) Delmarva Poultry Industry (DPI)-derived vaccine was characterized during serial passages in chicken embryo kidney (CEK) cells and after back-passage in embryonated chicken eggs (ECE) and in chickens. Both conventional and deep sequencing results consistently showed population changes occurred during adaptation to CEK cells. Specifically, thirteen amino acid (aa) positions seemed to be targets of selection when comparing the vaccine genome prior to and after 7 passages in CEK (CEKp7). Amino acid changes occurred at four positions in the S gene, and at two positions in the S gene large shifts in frequencies of aa encoded were observed. CEK adaptation shifted the virus population towards homogeneity in S. The changes achieved in the S1 gene in CEKp7 were maintained after a back-passage in ECE. Outside the S gene, amino acid changes at three positions and large shifts in frequencies at four positions were observed. Synonymous nucleotide changes and changes in non-coding regions of the genome were observed at eight genome positions. Inoculation of early CEK passages into chickens induced higher antibody levels and CEKp4 induced increased respiratory signs compared to CEKp7. From an applied perspective, the fact that CEK adaptation of embryo-attenuated Ark vaccines reduces population heterogeneity and that changes do not revert after one replication cycle in ECE or in chickens provides an opportunity to improve commercial ArkDPI-derived vaccines.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: A.M. Ghetas, G.E. Thaxton, C. Breedlove, V.L. van Santen, H. Toro. Effects of Adaptation of Infectious Bronchitis Virus Arkansas Attenuated Vaccine to Embryonic Kidney Cells. Avian Diseases (in press)

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Results have been submitted to the Avian Diseases journal. They are currently in press and will be published in the March 2014 issue of the journal. What do you plan to do during the next reporting period to accomplish the goals? Continue work with the NDV recombinant virus to provide cross protection among IBV strains.

Impacts
What was accomplished under these goals? Recombinant vaccines were developed. Specifically a recombinant adenovirus vaccine vectoring the S1 gene if infectious bronchitis virus was developed. The S1 gene sequence was synthesized based on the sequences predominant en the host. The vaccine protected chickens from homologous challenge. Resultsare in press in the Avian Diseases journal. Will be published in the March 2014 issue of this journal. A second vaccine was developed.A recombinant Newcastle disease virus vectoring the S2 gene of IBV proved to confer heterotypic protection against IBV when use in a prime and boost regime with the widely used Massachusetts attenuated strain. Results will also appear in the March 2014 issue of Avian Diseases.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2014 Citation: 1. Nichole Orr, Stephen L. Gulley, Haroldo Toro, Rodrigo Gallardo, Frederik W. van Ginkel. IgA as an Early Humoral Responder after Mucosal Avian Coronavirus Vaccination. Avian Diseases(in press). 2. Toro H, W Zhao, C Breedlove, Z Zhang, V van Santen, Q Yu (2013). Infectious Bronchitis Virus S2 Expressed from Recombinant Virus Confers Broad Protection against Challenge. Avian Diseases (in press). 3. Toro H, JF Zhang, R Gallardo, V van Santen, FW van Ginkel, K Joiner, C Breedlove (2013). S1 of Distinct IBV Population Expressed from Recombinant Adenovirus Confers Protection against Challenge. Avian Diseases (in press) 4. Ndegwa, EN, H Toro, VL van Santen. Comparison of vaccine subpopulation selection, viral loads, vaccine virus persistence in trachea and cloaca, and mucosal antibody responses after vaccination with two different ArkDPI-derived infectious bronchitis virus vaccines. Avian Diseases (in press)

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: We developed recombinant viruses encoding the spike (S1) proteins of IBV populations C2, C4, and C5, three of the at least five populations which become selected in chickens after attenuated ArkDPI vaccination (Gallardo et al., 2010). As expression model we used replication-defective recombinant adenovirus (Ad) vectors previously described (Toro et al., 2008; Tang et al., 2009). In brief, the complete S1 gene sequences of IBV populations C2, C4, and C5 (GB# EU359650, GQ484957, GQ484958) were optimized to the chicken codons and synthesized (Lochmuller et al., 1994; Shi et al., 2001). E1/E3-defective Ad-vectors encoding the codon-optimized S1 genes were constructed as described ((Toro et al., 2008;Tang et al., 2009). Groups of SPF chickens (n=12 to 18) were each primed IM at 3 days of age and boosted via the ocular route at 20 days of age with one of the Ad-constructs. The vaccination dose varied between 3.6 x 10^7 and 10^8 ifu/bird. Based on our previous studies, the chosen vaccine dosage was low and expected to provide 70% to 80% protection, thus allowing detection of differences in levels of protection. Additional experimental groups included unvaccinated and unvaccinated/unchallenged controls. All chicken groups except the unvaccinated/unchallenged control group were challenged on day 41 after hatch via eyes with 10^6.5 EID50/bird of an Ark serotype virulent strain (GB#JN861120). Evaluation of protection was assessed 4 days after challenge by clinical signs (incidence and severity of respiratory signs), concentration of IBV viral RNA in tears, and histopathological evaluation of the trachea. Challenged chicken groups showed differing levels of respiratory disease. Birds vaccinated with AdIBVS1.C2ch showed lower severity of respiratory signs compared to unvaccinated/challenged controls. However, AdIBVS1.C4ch and AdIBVS1.C5ch did not confer apparent protection against respiratory signs; chickens vaccinated with AdIBVS1.C4ch were indistinguishable from unvaccinated/challenged control chickens. Differences among groups in viral loads in tears measured by concentration of IBV viral RNA four days after challenge exhibited the same pattern as respiratory signs. The AdIBVS1.C2ch group exhibited a slight reduction in viral load compared to unvaccinated control chickens, and a significant reduction compared to AdIBVS1.C4ch vaccinated chickens. AdIBVS1.C5ch vaccinated chickens showed slightly less viral RNA than AdIBVS1.C4ch vaccinated chickens without achieving a significant difference. Blinded histopathological evaluation scored levels of necrosis, deciliation, and lymphocyte infiltration in the tracheal mucosa. Tracheal histopathology scores obtained by combining the scores of each chicken for necrosis, deciliation, and lymphocytic infiltration showed the exact same pattern when analyzed separately. The histopathological scores pattern of all groups was consistent with clinical signs and concentration of IBV viral RNA. Chickens vaccinated with AdIBVS1.C2ch showed significantly less (P<0.05) histological damage in the tracheal mucosa than unvaccinated controls and chickens vaccinated with the other constructs. PARTICIPANTS: We partnered with a vaccine developing company named Vaxin for the development of adenovirus constructs. The project provided training for veterinary student D. Pennington and graduate student Rodrigo Gallardo. TARGET AUDIENCES: Results were published in the refereed international Avian Diseases journal. Results were also published in the proceeding of an international meeting.Thus, we are targeting both a national and an international audience. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Results showed that the C2 recombinant construct was able to induce partial protection against challenge while neither the C5 nor C4 constructs provided protection, with the least protection provided by the C4 construct. The S1 amino acid sequence of C2 differs from the challenge strain at 17 positions. C4 differs from the S1 of the challenge strain at 18 positions and C5 at 23 positions. Thus C2, C4, and C5 have 96.9%, 96.7%, and 95.8% amino acid identity respectively to the S1 of the challenge strain. However, C4, which protected the most poorly, differs from C2, which provided the highest protection, only at amino acid position 56. At this position the highly hydrophilic asparagine [hydropathy (HP) index -3.5] of C2 has been replaced by the considerably less hydrophilic serine (HP index -0.8) in C4. This hydrophilicity difference likely changes the conformation of the epitope in C4. The fact that a change in one amino acid in this region significantly alters the induction of a protective immune response of the protein provides evidence that the first portion of S1 displays relevant immunoprotective epitopes. C5 differs from C2 at 6 positions including a change of histidine to tyrosine at position 43. Based on hydropathy this change could also alter the configuration of the protein considerably and might support the previous assumption. However, based on hydropathy C5 shows other important differences, for example at position 323 where the highly hydrophilic and positively charged arginine (HP index -4.5) in C2 is replaced by the considerably less hydrophilic and uncharged threonine (HP index -0.7). Interestingly AdC5 demonstrated slightly better protection than C4 in all measured parameters but because of other multiple differences no clear conclusions can be drawn at this time.

Publications

• Toro H, Gallardo R.A., van Santen, V.L., Zhang, J.F, Joiner, K.S. (2012). Development and evaluation of recombinant adenovirus encoding IBV S1 proteins of IBV ArkDPI populations selected in chickens.Proc VII. International Symposium Avian Corona- Metapneumoviruses & Complicating Pathogens.ISBN 978-3-9805907-9-2. Pages 225-229.

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: We have submitted preliminary results for publication in the peer-reviewed scientific journal Avian Diseases. PARTICIPANTS: Researchers Haroldo Toro, Vicky L. van Santen, Frederik W. van Ginkel, and Kellye S. Joiner of the Department of Pathobiology, Auburn University and JianFeng Zhang and Vaxin Inc., Birmingham, AL collaborated in this research. PhD graduate student Rodrigo A. Gallardo and summer scholar Daniela Pennington assisted the work. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We developed replication-defective recombinant adenovirus expressing codon-optimized IBV Ark S1 genes (AdArkIBV.S1ck). The S1 genes inserted in the Ad vector correspond to three different IBV populations (C2, C4, C5) that become selected in chickens after initial replication in the upper respiratory tract. The consensus sequence of the complete S1 of these predominant populations was optimized to the chicken codons and synthesized. The fragment containing the full-length synthetic S1 gene was inserted into the HindIII-XbaI site of the shuttle plasmid pAdHigh in the correct orientation under transcriptional control of the CMV major immediate-early promoter. Replication defective, E1/E3-defective Ad vectors encoding the codon-optimized S1 genes were constructed in PER.C6 cells. The AdArkIBV.S1ck vectors were validated by DNA sequencing. The seed virus was propagated in human 293 cells and titered[infectious units (ifu) per ml]. So far we have evaluate protection conferred by Ad vectoring the S1 of population named C2.In ovo vaccination with a low dose of AdArkIBV.S1ck resulted in a mild increase of systemic antibody and reduced viral shedding but no protection against IBV signs and lesions.

Publications

• Haroldo Toro, Daniela Pennington, Rodrigo A. Gallardo, Vicky L. van Santen, Frederik W. van Ginkel, JianFeng Zhang, Kellye S. Joiner. Infectious Bronchitis Virus Subpopulations in Vaccinated Chickens after Challenge. Avian Diseases (submitted for publication January 2012).

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Gallardo, R.A., V.L. van Santen, and H. Toro. Effects of CAV and/or IBDV on IBV Replication and Phenotypic Drift. American Association of Avian Pathologists Annual Meeting, Atlanta, GA, August 1-4, 2010. Gallardo, R.A., V.L. van Santen, F.J. Hoerr, and H. Toro. Effects of Infectious Bronchitis Virus on Chicken Testicles. (Poster) American Association of Avian Pathologists Annual Meeting, Atlanta, GA, August 1-4, 2010. PARTICIPANTS: Rodrigo Gallardo is a PhD student working with us on this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
1. Infectious bronchitis (IB) virus (IBV) continues to be the most common contributor to overall disease losses in poultry in the U.S. We have demonstrated high genetic and phenotypic heterogeneity within the virus populations of all available ArkDPI-derived attenuated vaccines. It has also become clear that the vaccine's predominant virus geno/phenotype is rapidly negatively selected during a single passage in chickens. We have demonstrated that the predominant geno/phenotype of IBV Ark vaccines further changes during host invasion, probably as result of distinct selective pressure in the microenvironment of the different tissues. 2. We assessed venereal transmission of IBV by artificially inseminating adult hens either with semen from IBV-infected roosters or with IBV spiked semen. IBV RNA was detected in the trachea of all hens inseminated with IBV-spiked semen and in 50% of hens inseminated with semen from IBV-infected males. The egg internal and external quality was negatively affected in hens inseminated with semen containing IBV. These results provide experimental evidence for IBV venereal transmission.

Publications

• Gallardo, R.A., F.J. Hoerr, W.D. Berry, V.L. van Santen, H. Toro (2011). Infectious Bronchitis Virus in Testicles and Venereal Transmission. Avian Diseases, (in press).
• Gallardo, R. A., V. L. van Santen, H. Toro (2010). Host Intraspatial Selection of Infectious Bronchitis Virus Populations. Avian Diseases 54: 807-813
• Toro, H. (2010) Infectious Bronchitis Virus: Dominance of ArkDPI-type Strains in the United States Broiler Industry during the Last Decade Brazilian Journal of Poultry Science 12: 79-86