WO2009143332A2

WO2009143332A2 - Poultry viral materials and methods related thereto

Info

Publication number: WO2009143332A2
Application number: PCT/US2009/044818
Authority: WO
Inventors: Holly S. Sellers Ph.D
Original assignee: University Of Georgia Research Foundation
Priority date: 2008-05-22
Filing date: 2009-05-21
Publication date: 2009-11-26

Description

POULTRY VIRAL MATERIALS AND METHODS RELATED

THERETO

FEDERAL FUNDING STATEMENT [0001] None.

RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application

Serial Number 61/055, 158, filed May 22, 2008.

FIELD OF THE INVENTION

[0003] The present invention relates to new materials and methods in the field of poultry virology, particularly in the field of the "infectious bronchitis virus," a virus that causes respiratory, reproductive and renal disease in all types of poultry. The invention is also in the field of spike glycoproteins and novel "Sl" portions of the spike glycoproteins, as well as nucleic acids which encode the proteins. Generically, the invention is in the field of veterinary molecular biology and virology.

BACKGROUND OF THE INVENTION

[0004] Infectious bronchitis virus (IBV) is a Group III Coronavirus, an RNA virus with "spike" protein antigens. IBV is the causative agent of an acute, highly contagious disease in chickens of all ages.

[0005] IBV changes rapidly in nature to yield variant viruses. Genetic changes are generally occur when two viruses infect the same cell, and sometimes result in a new serotype. Significant serotype-altered variants arise only periodically, and is suspected when vaccinated poultry flocks become symptomatic of the disease. The Sl subunit of the S glycoprotein is often investigated when such variation is discovered. Significant serotype variations in IBV have been characterized in 1962, 1987, 1992 and 1998-99. This application describes a significant variant from those.

[0006] IBV has been reported in all countries where an intensive poultry industry has been developed. The name "infectious bronchitis virus" is a misnomer, in that the pathogenic state is not limited to the respiratory tract; the liver, kidneys and reproductive organs are also damaged by the virus. Chickens up to four weeks of age are most susceptible to respiratory disease, infection leading to high rates of morbidity and to mortality resulting from secondary bacterial infection. Infection in layers results in a drop in egg production, or failure to lay eggs at full potential, together with an increase in the number of down-graded eggs with thin, misshapen, rough and soft-shells produced. Although layers usually recover from the disease, their egg production rarely returns to pre-infection levels. Thus, infection of flocks of chickens with IBV can have a serious economic effect.

[0007] The only practical means of preventing infectious bronchitis in poultry is to introduce low-virulence or non-virulent isolates, or other materials presenting immunologically-significant antigens. The poultry immune system reacts to both live attenuated and inactivated virus, although live attenuated virus is preferred in the market.

SUMMARY OF THE INVENTION

[0008] The present invention provides infectious bronchitis isolates having the serotypes and/or genotypes presented herein. In particular, those IBV isolates encoding or comprising an antigen described herein are provided.

[0009] More particularly provided are those IBV isolates, cells, amino acid sequences, vectors, vaccines, delivery devices or other biologicals comprising at least one amino acid herein, including those encoded by any one or more of the nucleic acids particularly disclosed herein. Modified amino acid sequences are also provided. [00010] The present invention also comprises nucleic acids which encode novel spike amino acid sequences or fragments thereof, especially those nucleic acid sequences which encode an amino acid sequence selected from the group consisting of: SEQ ID NO:4; SEQ ID NO:5; and SEQ ID NO: 6. The present invention also comprises nucleic acid sequences comprising nucleic acid sequences herein, especially those nucleic acids comprising SEQ ID NO:1 and/or SEQ ID NO:2 as well as nucleic acids which are at least 90% similar, preferably 95 to 99% similar, most preferably over 98% similar to SEQ ID NO:1 and/or SEQ ID NO:2, using BLAST analysis. The present invention also provides nucleic acid sequences comprising nucleic acid sequences herein, especially those nucleic acids comprising SEQ ID NO:3, as well as nucleic acids which are at least 87% similar, preferably 90 to 99% similar, most preferably over 95% similar to SEQ ID NO:3, using BLAST analysis. [00011] Nucleic acids which encode the sequences, as well as amino acid sequences which are encoded by these sequences are therefore also provided. In particular, the present invention provides nucleic acids comprising nucleic acids encoding amino acids 24-61, 132-149, and 291-398 of SEQ ID NO: 6, as well as amino acids comprising the corresponding amino acid sequences.

[00012] Also provided are amino acid sequences comprising amino acid sequences selected from the group consisting of: SEQ ID NO:4, SEQ ID NO:5; SEQ ID NO:6. However, any amino acid sequence comprising an amino acid sequence which is over 98% similar to SEQ ID NO:4 and/or SEQ ID NO:5, using BLAST analysis is preferred. Also preferred are any amino acid sequences comprising an amino acid sequence which is over 95% similar to SEQ ID NO:6, using BLAST analysis. [00013] Moreover, the present invention provides poultry virus isolates comprising the nucleic acids and/or the amino acids described herein. [00014] RNA sequences are particularly provided by the present invention.

Preferred are RNA sequences which comprise RNA sequences complementary to any of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. Positive strand RNA sequences which comprise at least one RNA sequence complementary to SEQ ID NO:3 are particularly preferred. Also provided are mRNA, ssRNA and dsRNA, preferably those which directly translate the present amino acid sequences via RNA polymerase. Preferred is a single stranded RNA that is positive in polarity, and comprises an RNA molecule that encodes an Sl glycoprotein subunit that is greater than: 94%, 95%, 96%, 97%, 98%, 99% or 100% similar to SEQ ID NO:3, using BLAST analysis. Most preferred is a positive polarity RNA molecule which comprises an RNA molecule that encodes SEQ ID NO:3.

[00015] The present invention also provides immunogens comprising the amino acids described herein.

[00016] Also provided are methods to introduce the present materials to at least one poultry, comprising: introducing a material of the present invention to a poultry's body part, wherein the body part is preferably selected from the group consisting of: eye; nose; ear; throat; skin. Preferred is a method to introduce at least one amino acid compound of the present invention to at least one poultry, comprising spraying the poultry with an isolate comprising at least one amino acid compound of the present invention. Most preferred is a method as described, wherein the isolate is a live, attenuated isolate. Also most preferred is spraying a lyophilized isolate as described, wherein the lyophilized isolate has been diluted in enough water to allow spraying at the concentration desired.

[00017] Moreover, the present invention provides useful materials for initiating immune response in poultry, especially providing vaccine materials. [00018] Moreover, methods for developing other materials using the compositions herein, including use in developing a live (naturally occurring low pathogenic) immunomodulator, attenuated and/or inactivated immunomodulator, whether alone or in combination (either with themselves or with other material) are also provided. [00019] In another embodiment, the present invention is a process for the preparation of live attenuated infectious bronchitis vaccine from a poultry virus isolate herein, comprising the steps of passaging a poultry virus isolate herein in a culture on a suitable medium for sufficient number of times to reduce its pathogenicity while retaining its immunogenicity, heat treating the passaged culture, harvesting the attenuated materials and processing the harvested material to produce a reduced immunological response, wherein the material is of the same Sl serotype described herein, or has the herein-described sequence identity with the present sequences. [00020] In another embodiment, a method of producing at least one poultry virus isolate comprising the steps of: inoculating a suitable substrate with at least one poultry virus isolate described herein; incubating the substrate; harvesting the resulting poultry virus isolate; and optionally attenuating the poultry virus isolate.

Definitions

[00021] "BLAST analysis" is intended to mean the nucleotide or protein sequence analysis program available from the United States National Center for Biotechnology

Information, using the broadest applicable search terms for that type of sequence, and using only those results available as of the filing date of this application.

[00022] "Poultry" is intended to embrace any breed of chicken, pheasant, emu, ostrich and other type of bird that is susceptible to infection by IBV. DETAILED DESCRIPTION OF THE INVENTION

[00023] The present invention provides useful poultry-associated materials which themselves can be used to further develop modifications of these poultry- associated materials.

[00024] For instance, the nucleic acids of the present invention may be used to produce constructs that express antigens. The antigens may be utilized to produce antibodies, which may be used for identifying field or laboratory isolates of the present invention.

[00025] Alternatively, the present isolates may be utilized to create antibodies, preferably monoclonal antibodies, reactive to the Sl glycoprotein herein, or portions thereof. Antibodies so created may then be used in screening assays, kits, research, and the like, as a method for identifying whether a poultry or a flock has been infected with a strain identical or similar to, the present isolates. In particular, monoclonal antibodies would be most useful in: research associated with the present isolates; confirming isolate introduction in poultry or flock; and identifying whether a poultry or flock has been infected with an identical serotype. Non-specific antibodies would be most useful in determining in determining whether a poultry or flock has been infected with a similar serotype. Raising antibodies is known in the art.

[00026] Moreover, the nucleic acids may be incorporated into a vector, attenuated live viral material of the same or different pathogen, and caused to be expressed in vitro or in vivo.

[00027] In addition, the nucleic acids of the present invention may be used to identify IBV isolates of the present invention, via hybridization, preferably under stringent conditions.

[00028] Moreover, virulent isolates of the present invention may also be attenuated, for the purpose of introducing to poultry while limiting virulence. The attenuation process is known in the art. For instance, the present invention may be passed through specific pathogen free (SPF) chicken embryos and heat treated to facilitate attenuation of the immunogenicity of the materials used for passage. Examples of such passages are described herein, and show that the isolates herein may be attenuated by the passage process.

[00029] Attenuated isolates are also obtainable by passaging virulent isolates of the present invention in a culture on a suitable medium a sufficient number of times to reduce its pathogenicity while retaining its immunogenicity. Preferably the avian viral material is passaged at least 20 times.

[00030] A preferred medium for such passaging is a Specific Pathogen Free embryonated egg. Inoculation of the eggs can be via the allantoic cavity, chorioallantoic membrane, yolk sac, amniotic cavity or even direct into the embryo. The virus can be passaged at regular intervals of from 7 hours up to 4 days. Commonly, passaging takes place between 16 to 36 hours, preferably every 24 hours. Alternatively, attenuation may also be achieved by passaging the isolate in avian cell culture, such as chick embryo kidney cells.

[00031] After the viral material is attenuated, the viral material may be stored until use. In an embodiment, the viral material is lyophilized. The lyophilized viral material may then be rehydrated for use. In another embodiment, the viral material is frozen.

[00032] Accordingly, the materials herein may be combined with pharmaceutically acceptable carriers or diluents. In certain embodiments, a stabilizer is optimally present. A viral isolate may be live, attenuated, or inactivated. Alternatively, the Sl subunit herein may be utilized after being either expressed or synthesized, or otherwise provided as an immunogen to susceptible poultry.

[00033] According to another embodiment of the invention, there is provided a process for the preparation of live avian viral material which comprises passaging the isolates described herein, in a culture on a suitable medium a sufficient number of times to reduce its pathogenicity while retaining its immunogenicity. Preferably the material is passaged at least five times.

[00034] Any material described herein is preferably administered by eye drop, nose drop, in drinking water, or by spraying the birds, at any age from one day old up to point of lay (about 18 weeks).

[00035] Moreover, any material herein may be combined with other viral or immunogenic material(s), for example Newcastle Disease Virus (NDV), Mareks Disease

Virus (MDV), Infectious Bursal Disease (IBD), reovirus, Egg Drop Syndrome 1976.

Avian Encephalomyelitis, Chicken Anemia Agent (CAA) and other IBV serotypes, for experimental or therapeutic use.

[00036] Alternatively, the isolates herein may be modified by use of enzymes or other molecular techniques so that it will not replicate in poultry if the poultry is subjected to it. [00037] The immunogens herein are not limited to the exact Sl glycoprotein enumerated; they include conservative substitutions of amino acids other than amino acids 24-61, 132-149, and 291-398 of SEQ ID NO:6.. The term "conservative substitution" refers to the replacement of an amino acid residue by a structurally similar residue. Examples of conservative substitutions include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like. [00038] Alternatively, the primers disclosed in the examples (SEQ ID NO:7 and

SEQ ID NO:8) can be used in PCR to amplify the Sl glycoprotein from a sample, and the products compared via BLAST analysis or hybridization, preferably BLAST analysis, but also hybridization under stringent conditions, to a nucleic acid product herein.

[00039] For all materials herein, preferred production is via growth in embryonated specific pathogen free (SPF) chicken eggs. However, the particular medium/substrate upon which the strain is grown is not substantially important to the invention. After harvesting, the viral material may be inactivated using, for example, formaldehyde or beta-propiolactone. Other acceptable substrates include, but are not limited to, mammalian cell lines, avian cell lines, and the like.

[00040] After any inactivation and, if necessary, adjusting of the pH and neutralising of the inactivating agent, the material may be mixed with an adjuvant. The adjuvant can be aluminum hydroxide or a composition consisting of mineral oil (e.g. Marcol 82) or a plant oil and one or more emulsifiers like Tween 80 and Span 80. [00041] Optionally, the present materials be administered by subcutaneous or intramuscular injection at between 1 to 20 weeks of age.

[00042] According to yet a further embodiment of the invention there is provided a method for reducing immunogenic response to IBV in birds, comprising administering the present materials, as hereinbefore described, to susceptible birds. [00043] While the invention has been described in connection with specific embodiments, it will be understood that it is capable of further modifications and the appended Claims are intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth whether now existing or after arising.

Example 1. Initial Isolate identification and characterization

[00044] Tracheas and kidneys from flocks in the field were obtained. Generally,

"GA07 IBV" was isolated from IBV-vaccinated flocks exhibiting severe mortality and flushing at approximately 28-35 days of age. "GA08 IBV" was isolated from IBV- vaccinated flocks wherein the primary clinical complaint was airsacculits either on the farm or at the processing plant, resulting in high condemnations.

[00045] Viral material isolation was performed in 9-11 day old specific pathogen free embryos inoculated by the chorioallantoic sac. At 48 hours post inoculation (embryo passage 1, El) , allantoic fluid was aseptically removed and tested for hemagglutination activity (HA) with 5% chicken red blood cells (CRBC). Additionally, allantoic fluid was treated with lOU/ml type V Neuraminidase and incubated at 37°C for 30 minutes. Following incubation, neuraminidase-treated allantoic fluid was tested for HA activity with 5% CRBCs. Neither untreated or treated allantoic fluid hemagglutinated CRBCs. Allantoic fluid from this passage was passed into a second set of 9-11 day-of embryonation SPFs embryos (embryo passage 2, E2). As with El, the E2 passage was tested for HA with CRBCs with and without neuraminidase treatment. The E2 passage was negative for HA. Passaging of the samples was carried out to E5 for GA07 isolate (60173) and to E4 for the GA08 isolate (64513). Embryo viability was examined on a daily basis and mortality documented. In addition, at 7 days post inoculation, all embryo passages were opened and evaluated for IBV-specific lesions such as embryo stunting, curled toes, clubbed down, presence of kidney urates. Suspect infectious bronchitis virus isolation was based on embryo lesions and mortality patterns which were consistent with IBV. Confirmation and characterization of IBV isolates was performed using RT-PCR of a region of the spike glycoprotein, Sl subunit.

Example 2. Genetic characterization of GA07 and GA08 IBVs.

[00046] Allantoic fluid from embryo passages consistent with IBV isolation were further tested for EBV by RT-PCR of the Sl gene. Primers used for amplification (designated LC - IBV LC forward - 5' ACT GGC AAT TTT TTC AGA - 3' (SEQ ID NO: 7) and IBV LC reverse 5' ACA GAT TGC TTG CAA CCA C 3' (SEQ ID NO:8) ) have been shown to amplify the Sl gene between hypervariable region 1 and 2, yielding an intended product of 348 bp. Additionally, an 800 bp product (not the intended 348bp) was observed for several isolates due to the phenomenon of mispriming downstream of intended reverse primer location which has been observed on occasion. Sequence analysis of either product confirmed presence of IBV nucleic acid in allantoic fluid from suspect embryos, hi addition, BLAST analysis of GA07 IBV isolates with IBV Sl sequences available on GenBank revealed the highest similarity of nucleotide sequence (at 86-88%) to a nephropathogenic IBV isolated in Australia. Similarity to current U.S. commercial vaccines (at the nucleotide level) was between 80-83%. Sequence analysis of the nucleotide sequence of GA08 isolates with public domain sequences resulted in an 85—88% similarity to a variant IBV isolate called CU84074. Multiple GA07 and GA08 isolates were taken from multiple flocks, with phylogenetic analysis revealing a high degree of similarity within each GA07 and GA08 group (98-100%). The isolation of these variant strains of IBV from multiple flocks of chickens are not similar to endemic strains of IBV in the U.S. It has been well established that the sequence of the Sl gene (nucleotide and amino acid) correlates well with serotype.

Example 3. Attenuation of IBV isolates.

[00047] While many isolates within each group (GA07 and GA08) were identified according to the processes herein, representative isolates were selected for attenuation (continued passage to reduce pathogenicity) in chicken embryos for a live virus vaccine master seed production.

[00048] The embryo passage for isolates was continued in successive embryo passages in 9-11 day old embryos inoculated via the chorioallantoic sac (CAS) route of inoculation. Allantoic fluid was harvested between 32-36 hours post-inoculation and subsequently passed in 9-11 day old embryos as before. Every 10^th passage, additional embryos were inoculated and incubated out to the 7^th day post inoculation and at that time, opened for visual examination of embryos. Additionally, allantoic fluid from every 10^th passage was tested by RT-PCR of the IBV Sl LC region followed by sequence analysis to confirm the presence of the original IBV isolate. At the 50^th embryo passage, allantoic fluid was collected and saved for further passage as well as, titrated in 9-1 1 day old embryos, viral titer was determined using the method of Reed and Meunch resulting in representation of virus concentration in embryo infective dose 50 (EID50). Titrated viruses was safety tested in 2 week-old chickens. Two week old broilers or SPF chickens were challenged according to procedures outlined in the 9 CFR (Code of Federal Regulations). When the embryo passage that was considered attenuated had been safety tested, viral stock was then tested for extraneous viral and bacterial agents, propagated as before, and titrated in chicken embryos.

Example 4. Backpassage in chicks.

[00049] The attenuated GA08 IBV was backpassaged in commercial broiler chicks between 1-5 days of age (See table 1). Day-old, nonvaccinated commercial broilers were obtained. For the BP#1, ten one-day-old chicks were inoculated via eye drop with the attenuated GA08 at 10⁴ EID50/bird. The negative control group (n=10) was inoculated with lOOul of sterile PBS. At 5 days post inoculation, tracheal swabs were collected from each bird and pooled (by treatment group) in sterile PBS. Birds were euthanized and tracheas harvested and fixed in formalin per group. For BP# 2-10, 50 ul of swab supernatant from 1) GA08 and 2) negative controls was used to inoculate the next group of chicks via the trachea. At 5 days post inoculation, tracheal swabs were collected from each bird and pooled (by treatment group) in sterile PBS. Birds were euthanized and tracheas harvested and fixed in formalin by group. [00050] Histological scoring on cross and longitudinal trachea sections was performed.. Routine IBV PCR was used to evaluate tracheal swabs from GA08 groups at each backpassage.

[00051] Sequencing of IBV PCR products was performed on Backpassages #1 and

#5. No clinical signs or significant macroscopic lesions were observed in any of the backpassages #1-10 at 5 days post inoculation. No significant difference in tracheal lesion scores were observed between vaccinates and negative controls. Tracheal lesion scores were mild (<2.2) in all groups at each backpassage. IBV was detected by RT- PCR in swabs from each GA08 group in BP#l-6. No IBV was detected in tracheal swabs from BP#7-10. The GA08 live attenuated vaccine (E71) is stably attenuated and does not pose the risk of becoming more virulent throughout the course of 10 backpassages in susceptible chicks. In addition, replicating virus was not detected beyond the 6^th backpassage. Example 5. IBV protection in commercial broilers vaccinated at day-of-hatch with NDV C2-GA08 IBV- Ark IBV.

[00052] The treatment groups are outlined in Table 1.

Table 1. Group identification and treatment designations at day-of-hatch and 14 days-of-age.

[00053] Two groups of day-of-hatch commercial broilers were delivered to the research facility. One group contained 30 chicks that were spray vaccinated in the hatchery with a combination of live C2NDV -GA08IBV-Ark IBV. This group was divided into 3 groups of 10 birds each and placed into Horsfall Bauer forced air, positive pressure (FAPP) isolation units. A second group contained non-vaccinated chicks and 10 were placed into Horsfall Bauer forced air, positive pressure (FAPP) isolation units. Birds were given food and water ad libitum throughout the trial. At 14 days-of-age, birds in groups 2-4 were challenged with either 10^{4 5} EID₅₀ GA08 IBV or 10⁴ ° EID₅₀ Ark IBV (see Table 1) via eye drop. Five days post-challenge (5 dpc), the birds were evaluated for clinical signs and necropsied.

[00054] On the day of necropsy (5 dpc), clinical signs were evaluated in all birds

(See Figure 1). The birds were bled for IDEXX IBV ELISA (See Table 2) and tracheal swabs were taken for IBV real time RT-PCR. Following euthanasia, cross and longitudinal trachea sections (below the point of swabbing) were collected, fixed in neutral buffered formalin, and histologically evaluated by Dr. Susan Williams. Histological scoring on was performed by Dr. Susan Williams Results are summarized below. [00055] Table 2. IBV ELISA results from serum collected 5 days post challenge

(19 days of age).

Ten serum samples were collected from each group.

[00056] All groups were negative for IBV antibodies at 19 days of age as evaluated by IBV ELISA. Clinical signs observed in the vaccinated/unchallenged group were mild and limited to conjunctivitis and airsacculitis in 2/10 birds. In addition, mean tracheal lesion scores in this group were less than the other 3 treatment groups and considered normal for birds this age reared in isolation units. IBV was not detected from the tracheal scrapings in the vaccinated/unchallenged group by real time RT-PCR. Clinical signs were observed in all birds from the unvaccinated/GA08 challenge group. This group served as the positive challenge control group. Moderate to severe airsacculitis was observed in 10/10 birds. Despite the fact that the mean tracheal lesions scores in this group (ring and longitudinal) were not significantly higher than either vaccinated/challenged group, the histological lesions were more severe. IBV was detected from the tracheal scrapings in the unvaccinated/GA08 challenge group. The quantity of viral genome was slightly less than both vaccinated/challenge groups. Clinical signs were observed in both vaccinated/GA08 challenged groups. The airsacculitis observed in 4/10 (VX/GA08 Chall) and 6/10 (VX/Ark Chall) was mild and not as severe as the UnVX/GA08 Challenged group. The tracheal lesions scores for all GA08 challenged groups were numerically similar however, both vaccinated groups had less severe lesions than in the UnVx/GA08 chall group. IBV was detected equally from both vaccinated/GA08 challenged groups.

Claims

We claim:

1. A composition of matter comprising an Sl glycoprotein subunit encoded by a nucleic acid having at least 90% similarity to SEQ ID NO:3, using BLAST analysis.

2. A composition of matter of claim 1, wherein said nucleic acid has at least 95% similarity to SEQ ED NO:3, using BLAST analysis.

3. A composition of claim 1, wherein the material is an attenuated infectious bronchitis viral isolate.

4. A composition of claim 3, wherein the material is lyophilized.

5. A composition of claim 4, wherein the material is diluted in liquid.

6. A composition of matter comprising a nucleic acid which encodes an amino acid sequence selected from the group consisting of: SEQ ID NO:4; SEQ ID NO:5; and SEQ ED NO: 6.

7. A nucleic acid comprising a nucleic acid having at least 90% similarity to SEQ ID NO:3, using BLAST analysis.

8. A nucleic acid of claim 7, wherein said nucleic acid has at least 95% similarity to SEQ ED NO:3, using BLAST analysis.

9. A composition of claim 8, which is an attenuated infectious bronchitis viral isolate.

10. A nucleic acid comprising a nucleic acid having at least 95 similarity, using BLAST analysis, to a sequence selected from the group consisting of SEQ ED NO:2 and SEQ ED NO:3

11. A method for introducing infectious poultry virus S 1 glycoprotein to a poultry comprising introducing a composition of claim 1 to a poultry's body.

12. A method for introducing infectious poultry virus Sl glycoprotein to a poultry comprising introducing a composition of claim 7 to a poultry's body.

13. A method of claim 12, wherein said composition is an attenuated infectious bronchitis viral isolate.

14. A method of claim 13, wherein said composition is lyophilized, diluted in liquid, and introduced to said body by spraying.

15. A method of claim 12, wherein said composition is co-introduced with other viral material.

16. A method of producing at least one poultry infectious bronchitis virus isolate comprising the steps of: identifying, in a nucleic acid sample obtained from poultry having infectious bronchitis, whether the nucleic acid of SEQ ID NO:3 is at least 90% similar, using BLAST analysis.

17. A method for reducing immunogenic response to infectious bronchitis virus in poultry susceptible to infectious bronchitis virus, comprising introducing a composition of claim 3 to a poultry susceptible to infectious bronchitis virus.

18. A method for reducing immunogenic response to infectious bronchitis virus in poultry susceptible to infectious bronchitis virus, comprising introducing a composition of claim 7 to a poultry susceptible to infectious bronchitis virus.

19. A method for reducing immunogenic response to infectious bronchitis virus in poultry susceptible to infectious bronchitis virus, comprising introducing a composition of claim 9 to a poultry susceptible to infectious bronchitis virus.

20. A monoclonal antibody that binds to SEQ ID NO:3.