Abstract
In the present study we describe the rapid development of an attenuated live vaccine for GA08, a new serotype of infectious bronchitis virus, using a heat-treatment method. Incubation of the GA08 strain of IBV at 56°C and passage in embryonated eggs was used as a method to fast track the attenuation process. The virus was incubated in a 56°C water bath and aliquots were removed every 5 min for up to 1 h, and then each aliquot was inoculated into 10-day-old embryonated eggs. Virus with the longest incubation time that produced lesions in the embryos was harvested, again incubated at 56°C as described and passaged in embryonated eggs. Attenuation of the virus, designated GA08/GA08HSp16/08, was verified in 1-day-old specific pathogen free chicks. A 10x dose of the vaccine was found to be safe for 2-week-old broiler chicks of commercial origin. The efficacy of the heat-treated attenuated virus was determined by vaccinating broiler chicks of commercial origin at 1 and 14 days of age intraocularly/intranasally. Vaccinated birds that were challenged with 104.5 median embryo infectious doses of pathogenic GA08 virus/bird at 28 days of age were protected from the disease, and challenge virus was only detected in the trachea of one of 21 birds by real-time reverse transcriptase-polymerase chain reaction at 5 days post challenge. The attenuation process took 10 weeks to complete, which is a substantially shorter time than required to attenuate infectious bronchitis virus by serial passage in embryonated eggs without heat treatment (38 weeks or more).
Introduction
Infectious bronchitis virus (IBV) is a group 3 avian coronavirus that causes a highly infectious upper respiratory tract disease in chickens, as well as decreased egg production and egg and eggshell quality in hens; some strains can cause nephritis. It is worldwide in distribution, and similar group 3 coronaviruses have been isolated from many wild bird species, as well as from a beluga whale (Delphinapterus leucas) (Cavanagh et al., Citation2002; Jonassen et al., Citation2005; Liu et al., Citation2005; Mihindukulasuriya et al., Citation2008; Hughes et al., Citation2009; Muradrasoli et al., Citation2009; Woo et al., Citation2009). The enveloped virus has a single-strand positive-sense RNA genome that codes for the viral RNA-dependent RNA polymerase, four structural proteins (spike, envelope, membrane, and nucleocapsid) and numerous regulatory proteins (Masters, Citation2006). The spike (S) glycoprotein, which mediates cell attachment and virus–cell membrane fusion, and plays an important role in host cell specificity, forms club-shaped projections on the surface of the virus and consists of S1 and S2 subunits (Masters, Citation2006; Cavanagh & Gelb Jr, Citation2008). Control of the disease is extremely important because IBV predisposes birds to potentially lethal secondary pathogens (Cavanagh & Gelb Jr, Citation2008). Attenuated live vaccines and killed vaccines are used in an attempt to prevent the disease. However, extensive genetic diversity and a high mutation rate result in many different types of the virus that do not serologically cross-react, making it important to vaccinate chickens with the type of IBV causing the disease (Cavanagh & Gelb Jr, Citation2008). There are approximately six different serotypes of IBV vaccine used in the USA, and although two or more vaccine types can be used to provide broad protection, the emergence of new variant viruses capable of causing disease in the vaccinated birds does occur.
Identifying the type of IBV causing disease in commercial chickens is the first step in controlling this highly infectious virus, but it is of little value if commercially available vaccines do not protect against it. Live IBV vaccines are produced by multiple passages in embryonated eggs, but that methodology can take months because typically 75 to over 100 passages are necessary to attenuate the virus. That combined with the time required to obtain licensing can postpone a new vaccine reaching the market for 18 months to 2 years or longer, and there is no guarantee that the variant virus will still be endemic at that time. Methodology that can shorten the time between IBV variant discovery and commercialization of a new vaccine may make it more attractive for vaccine companies to pursue development of a new IBV vaccine. Of course it is important to determine that the IBV variant is widespread and that a new vaccine is necessary for control.
In January 2008, a new IBV variant was first detected in a flock of 48-day-old broilers in northern Georgia and has since been isolated from chickens with respiratory disease in Georgia and South Carolina. Clinical signs and lesions associated with this virus, designated GA08, are generally mild and consist of conjunctivitis, mild tracheal râles, tracheitis and abdominal airsacculitis. Commercially available live IBV vaccines, either alone or in combination, did not provide protection against GA08 (unpublished data). In the present study, we describe a rapid heat-treatment attenuation process for the GA08 strain of IBV as well as safety and efficacy testing to examine the utility of the vaccine. Attenuation of the GA08/pass4/08 strain of IBV by heat treatment follows a protocol similar to the method used to attenuate the JMK and H strains of IBV (unpublished data). But it should be noted that IBV isolates can be vastly different with respect to growth characteristics and resistance to environmental conditions and this attenuation process may not work for all IBV types.
Materials and Methods
Viruses
The GA08/pass4/08 strain of IBV (titre 105.5 median embryo infectious dose [EID50] /ml) was used as the starting material for the heat treatment, as well as for challenge.
Heat-treatment attenuation
The GA08/pass4/08 virus was incubated at 56°C and 1 ml aliquots were removed every 5 min for 60 min. Each aliquot was inoculated (0.1 ml/egg) into the chorioallantoic sac of at least five 10-day-old embryonated chicken eggs and was incubated for 6 days. The embryos were examined and allantoic fluid was harvested from the eggs inoculated with the longest heat treatment that induced lesions in the embryos. That allantoic fluid was then used in a subsequent round of heat treatment followed by inoculation into embryonated eggs. The procedure was repeated eight times. Virus harvested from the last heat treatment was passaged four additional times in 10-day-old embryonated eggs (allantoic fluid was harvested at 48 h post inoculation) without heat treatment to increase the volume and titre of the virus. Following each heat-treatment passage, the allantoic fluid used for the subsequent passage was examined for the presence of virus by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) as previously described (Callison et al., Citation2006). The titre of the 16th passage (four initial passages, plus eight heat-treatment passages, plus four additional passages) of the virus designated GA08/HSp16/08 was determined in 10-day-old embryonated eggs and the titre was calculated by the method of Reed and Muench as described previously (Gelb & Jackwood, Citation2008; Villegas, Citation2008).
Purity and virus identity tests were conducted as described in Section 113.300 of Title 9 of the Code of Federal Regulations (USDA, Citation1999). Passage 16 of the GA08/HSp16/08 virus was tested for freedom from bacteria, fungi, mycoplasma, and extraneous viruses, including chicken anaemia virus, haemagglutinating viruses and avian leucosis virus (Dufour-Zavala et al., Citation2008). The virus was also tested for attenuation in 1-day-old specified pathogen free (SPF) Leghorn chicks (Charles River SPAFAS, N. Franklin, Connecticut, USA) according to the procedures in Section 113.327 of Title 9 of the Code of Federal Regulations (USDA, Citation1999). Chicks were randomly divided into two groups of 10 birds each and housed in positive-pressure Horsfall isolation units. Feed and water were given ad libitum and the birds were examined twice daily. Birds in the first group were given 104 EID50/bird of pass 16 of the GA08/HSp16/08 virus by eye drop and intranasally. This dose was selected because, in our experience, it is an amount of virus likely to infect and produce disease. The safety of the virus was evaluated in a separate experiment using a 10x dose (see Safety testing below). The second group of birds, which served as a positive control, was given 104 EID50/bird of pass 16 of the virus without heat treatment. The third group of birds was not exposed and served as a negative control. Five birds in each group were killed humanely and examined post mortem at 5 and 10 days post exposure. Tracheal swabs were placed in 1 ml ice-cold phosphate-buffered saline (pH 7.4) and stored at –80°C until tested for the presence of viral RNA by real-time RT-PCR. The lower portion of the trachea (below the swabbed area) was fixed in 10% neutral buffered formalin, routinely processed and embedded into paraffin blocks. Thin sections were cut and stained with haematoxylin and eosin and examined by light microscopy. Epithelial hyperplasia, lymphocyte infiltration and the severity of epithelial deciliation were scored for each trachea from 1 to 4 (1=normal, 2=focal, 3=multifocal and 4=diffuse). The least-significant difference of the means was statistically calculated with the Student t test for each pair using JMP Statistical Discovery Software (SAS Institute Inc., Cary, North Carolina, USA) as previously described (Jackwood et al., Citation2003).
Safety testing
Twenty-five commercial broiler chicks (Fieldale Farms, Baldwin, Georgia, USA) were housed in positive-pressure Horsfall isolation units, given feed and water ad libitum and used to test the safety of the GA08/HSp16/08 virus (pass 16) according to Section 113.327, d, 2, of Title 9 of the Code of Federal Regulations (USDA, Citation1999). A 10x dose (105 EID50) of virus was given by eye drop to the chicks at 5 days of age and the birds were examined for clinical signs twice daily for 21 days. Five additional birds were maintained as negative controls. Prior to exposure, sera were collected from seven birds and stored at –20°C until tested for maternal antibodies by commercial enzyme-linked immunosorbent assay (ELISA) (IDEXX, Westbrook, Maine, USA). In this ELISA, titres >256 are considered positive. At the end of the experiment, sera were collected and stored at –20°C until tested by ELISA (IDEXX) when the birds were killed and tracheas collected and processed for histopathology as described above.
Efficacy testing
Thirty-three 1-day-old commercial broilers (Fieldale Farms) were randomly divided into three groups. Twenty-one birds in the first group were vaccinated intraocularly and intranasally (104 EID50 /bird) with the GA08/HSp16/08 virus at 1 and 14 days of age. Six birds each in the second and third groups were not vaccinated. The first and second groups of birds were challenged intranasally (104.5 EID50/ bird) with pathogenic GA08/pass4/08 at 35 days of age. At 5 days post challenge, all birds were killed and examined post mortem. At this time, tracheal swabs and sera were collected and stored as described above. Efficacy was based on not less than 90% of the controls being positive for virus recovery and not less than 90% of the vaccinates negative for virus recovery. The lower halves of tracheas were collected and processed for histopathology as described above. Scoring and statistical analysis were also as described above.
RNA extraction and real-time RT-PCR
Viral RNA was extracted from 50 µl phosphate-buffered saline from the tracheal swab using the MagMAX-96 RNA Isolation Kit (Ambion Inc., Austin, Texas, USA) according to the manufacturer's protocol on a KingFisher magnetic particle processor (Thermo Scientific, Waltham, Massachusetts, USA). Real-time RT-PCR was conducted using a Smart Cycler II (Cepheid, Sunnyvale, California, USA) and the AgPath-ID™ One-Step RT-PCR kit (Ambion Inc.) according to the manufacturer's recommendations. Primers and probes for the real-time RT-PCR were previously published (Callison et al., Citation2006) and consist of a forward primer IBV5′GU391 (5′-GCT TTT GAG CCT AGC GTT-3′), a reverse primer IBV5′GL533 (5′-GCC ATG TTG TCA CTG TCT ATT G-3′) and a Taqman® dual-labelled IBV5′G probe (5′-FAM-CAC CAC CAG AAC CTG TCA CCT C-BHQ1-3′). The primers were obtained from Integrated DNA Technologies (Coralville, Iowa, USA) and the Taqman® probe was synthesized by BioSearch Technologies (Novato, California, USA). Real-time RT-PCR components and thermocycler parameters were conducted as previously described, and a standard curve for the assay, which was previously published, was used to calculate the approximate genome copy number for each sample (Callison et al., Citation2006). Any sample below the calculated level of detection (100 genome copies) was considered negative.
Negative control swab samples, which were swabs placed in buffer at post-mortem examination, were included in the RNA extraction and real-time RT-PCR assay. In addition, known negative and positive samples for RNA isolation and for real-time RT-PCR were included at every 10th sample analysed. Negative controls also included allantoic fluid collected from non-inoculated SPF eggs, and the isolated material was carried forward to the real-time RT-PCR assay as well as real-time RT-PCR assay reaction mixture without RNA template. Positive controls consisting of allantoic fluid containing Massachusetts (Mass) 41 type IBV whole virus (104.0 EID50/ml) was included for the RNA extraction and carried forward to the real-time RT-PCR-positive (from previous real-time RT-PCR-positive samples) IBV RNA from the Mass 41 strain of IBV, which was used as a template in the real-time RT-PCR assay. If any of the controls were not as expected, the experimental samples and controls were retested.
Molecular characterization
Viral RNA was extracted from 50 µl allantoic fluid containing virus using the MagMAX-96 RNA Isolation Kit (Ambion Inc.) according to the manufacturer's protocol on a KingFisher magnetic particle processor (Thermo Scientific). The S1 glycoprotein gene was RT-PCR amplified from the extracted RNA following previously published methods, and the amplified product was sequenced (Lee et al., Citation2000, Citation2003). The amplified products were purified using GenElute™ spin columns (Supelco, Bellefonte, Pennsylvania, USA) and concentrated using Microcon™ 30 columns (Amicon, Beverly, Masachusetts, USA). The 3′ primer designated Degenerate 3′ (5′-CCATAAGTAACATAAGGRCRA-3′) and a 5′ primer designated NEWS1OLIGO5′ (5′-TGAAACTGAACAAAAGAC-3′) were previously published (Lee et al., Citation2000). Sequencing was conducted at the Molecular Genetics Instrumentation Facility (University of Georgia, Athens, Georgia, USA) with the Prism™ DyeDeoxy terminator cycle sequencing kit according to the manufacturer's recommendations (Perkin Elmer, Foster City, California, USA).
Sequences were compared by nucleotide–nucleotide BLASTn and protein–protein BLASTp search analyses online at the National Center of Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/). Sequences identified by BLAST analysis as well as previously published IBV vaccine sequences (McKinley et al., Citation2008) were used for ClustalW alignment (MegAlign software version 1.03; DNASTAR Inc., Madison, Wisconsin, USA) and phylogenetic trees were constructed with the neighbour-joining method, the maximum parsimony method, and UPGMA with 1000 bootstrap replicates (MEGA4, http://www.megasoftware.net/index.html) (Tamura et al., Citation2007).
Results
Attenuation
Data on the heat-treatment attenuation of GA08/pass4/08 are presented in . The time of heat-treatment at 56°C ranged from 10 to 55 min and did not appear to show a relationship with passage number or virus titre as determined by real-time RT-PCR. Passage 13 of the virus in embryonated eggs was conducted without prior heat treatment, and the titre of the virus as determined in embryonated eggs was 104.45 EID50/ml. To increase the titre, the virus was passaged four more times (virus containing allantoic fluid was harvested at 48 h post inoculation) in embryonated eggs without prior heat treatment and the titre of the 16th embryo passage designated GA08/HSp16/08 was determined to be 106.63 EID50/ml. The 16th embryo passage of GA08/HSp16/08 was negative for bacteria, fungi, mycoplasma, chicken anaemia virus, haemagglutinating viruses and avian leucosis virus, and was used in subsequent safety and efficacy experiments.
Table 1. Heat treatmenta attenuation of IBV GA08/pass4/08.
No clinical signs were observed in 1-day-old SPF Leghorn chicks given 104 EID50/bird by eye drop and intranasally at either 5 or 10 days post inoculation (). In addition, the average tracheal lesion scores (1.0) from birds on both days were identical to the negative control birds (). In contrast, all the birds given pass 16 of the virus without heat treatment had clinical signs, challenge virus was detected in all of the birds and tracheal lesions (average score 2.95) indicated all of the birds had acute tracheitis.
Table 2. Attenuation testing of IBV GA08/HSp16/08 in 1-day-old SPF chicks.
Safety testing
Twenty-five commercial broiler chicks were given 105 EID50/bird (10x dose) of the GA08/HSp16/08 virus at 5 days of age and no clinical signs were observed for 21 days, when the birds were killed and examined post mortem. In addition, no clinical signs were observed in five additional birds maintained as negative controls at the same time. Maternal antibody titres were detected in sera collected prior to treatment (at 5 days of age) with six out of seven birds positive and a geometric mean ELISA titre of 588. No antibody titres were observed in the control birds at the time of killing. Sera collected from the treated birds 21 days after exposure and tested by ELISA resulted in five out of 25 birds being positive with a geometric mean ± standard deviation titre of 866.9 ± 831.9 for the positive birds only. Tracheas collected 21 days after infection and processed for histopathology showed no microscopic lesions (score=1.0) in the treated birds as well as the negative controls.
Efficacy testing
The efficacy testing data are presented in . None of the broilers vaccinated intraocularly and intranasally at 1 and 14 days of age with 104 EID50/bird of the GA08/HSp16/08 virus and challenged with GA08/pass4/08 at 35 days of age had clinical signs of disease at 5 days post challenge. Six birds not vaccinated and challenged intranasally (104.5 EID50/ bird) with GA08/pass4/08 at 35 days of age had clinical signs consisting of watery eyes, nasal mucus and tracheal râles at 5 days post challenge. No clinical signs were observed in negative control birds 5 days post challenge. Challenge virus was detected in tracheal swabs by real-time RT-PCR in one bird in the vaccinated and challenged group when examined post mortem, whereas virus was detected in all six birds in the non-vaccinated and challenged group, indicating that our time point of 5 days post challenge for virus detection was suitable. The tracheal lesion scores for the vaccinated and challenged birds were statistically higher than the negative controls but not as high as the non-vaccinated challenged group (P<0.05), indicating that some virus replication occurred in the challenged birds.
Table 3. Efficacy testing of the GA08/HSp16/08 virus in commercial broiler chickens at day 5 following challenge at 35 days of age with GA08/pass4/08.
No serum antibody titres were detected by ELISA in the negative control birds or the challenge control birds (). Seventeen out of 21 vaccinated and challenged birds were positive for IBV antibodies with a geometric mean antibody titre by ELISA of 590. The antibody titres ranged from 462 to 2691.
Molecular characterization
The S1 subunit of the spike gene for the GA08/pass4/08 challenge strain, for the GA08/08/08 pass 16 non-heat-treated strain and for the GA08/HSp16/08 the heat-treated strain were sequenced, and sequences were submitted to GenBank (accession numbers GU361606, GU734804 and GU360617, respectively). ClustalW was used to align the S1 protein sequences, and phylogenetic analysis computed using the neighbour-joining method and the Nei–Gojobori method for the GA08 isolate from the index case GA08/GU301925/08 (GenBank accession number GU301925), GA08/pass4/08, GA08/08/08 strain passage 16 and GA08/HSp16/08 showed the sequences to be from 91.5 to 96.9% similar. The phylogenetic reconstruction with other representative IBV strains is presented in . The GA08 viruses cluster in a distinct group. They border the group containing HN99, JAAS/04 and N1/62 with 80.4 to 85.6% similarity (). They are also adjacent to the group of California IBV isolates CA/557/03, CA/CA12495/98, CA/12495/98 and CAV/CAV9437/95, with 78.7 to 83.5% sequence similarities, as well as Ark/ArkDPI/81 with 77.8 to 82.5% similarity ().
Figure 1. Phylogenetic tree showing amino acid sequence relatedness of S1 proteins computed using neighbour-joining and Nei–Gojobori methods with 1000 bootstrap replicates. The amino acid sequences were aligned with ClustalW (MEGA 4.0.2; Tamura et al., 2007) and the amino acid substitutions (x100) are shown. GenBank accession numbers: CAV/1686/95=AF027511, CAV/CAV9437/95=AF027510, CAV/CAV56b/91=AF027509, CA/CA12495/98=AF520604, CA/557/03=DQ912828, HN99=AY775551, JAAS/04=AY839140, N1/62=U29522, GA08/HSp16/08=GU361607, GA08/08/08 pass 16= GU734804, GA08/pass 4 challenge strain= GU361606, GA08/S1/GU301925=GU301925, Ark/Ark99/73=L10384, Ark/ArkDPI/81=AF006624, PP14/PP14/93=M99483, CAL99/CA1535/99=DQ912831, CAL99/NE15172/95=DQ912832, Holte/Holte/54=L18988, JMK/JMK/64=L14070, Gray/Gray/60=L14069, SE17/SE17/93=M99484, Iowa/Iowa609/56=GU361608, B/D207/84=X58003, B/D274/84=X15832, B/UK167/84=X58065, B/UK142/86=X58066, E/D3896/84=X52084, CAV/CA1737/04=DQ912830, DMV/5642/06=EU694402, QX/IBVQX/99=AF193423, 793B/4-91/91=Z83975, Mass/H52=AF352315, Mass/H120=EU822341, Mass/Mass41/41=AY561711, Mass/Beaudette=M95169, Conn/Conn46/51=L18990, FL/FL18288/71=AF027512, DE/DE072/92=U77298, GA98/CWL470/98=AF274437, Dutch/D1466/81=M21971.
Table 4. Sequence distances (percentage identity) of S1 protein alignment (ClustalW).
Discussion
In the present study we report a heat-treatment method that was used to shorten the time required to attenuate the GA08 strain of IBV. The GA08 heat-treated virus was tested for attenuation, safety and efficacy. Attenuation of GA08/pass4/08 in 1-day-old chicks was accomplished by exposure of the virus to 56°C followed by propagation in embryonated eggs eight times (pass 5 to pass 12). Because the titre of the virus was low (104.45/ml) it was passaged in embryonated eggs four more times (to pass 16) to increase the titre for use in safety and efficacy studies. IBV is heat labile, being inactivated at 56°C for 15 min (Cavanagh & Gelb Jr, Citation2008). From one passage to the next, the longest 56°C incubation time that did not completely inactivate the virus varied from 15 to 55 min. The incubation times did not appear to correlate with the virus titre. Studies on severe acute respiratory syndrome coronavirus (SARS-CoV) showed that incubating the virus at 56°C for 60 min or longer reduced the titre to undetectable levels (Kariwa et al., Citation2006). However, coronavirus inactivation is apparently dependent on the amount of protein in the sample. Addition of protein to 20% of a sample containing SARS-CoV resulted in infectious virus following heat treatment at 56°C for 60 min (Rabenau et al., Citation2005). The protein content of egg albumen is approximately 10% (Stadelman & Cotterill, Citation1977), which may account for the presence of live IBV following incubation at 56°C for over 15 min (see ).
Based on these data, it would appear that IBV subpopulations resistant to heat inactivation are less virulent for chickens. The mechanism of action of heat-treatment attenuation is unknown, but heat inactivation of coronaviruses is thought to be through disruption of the virus structure. Stability studies on the SARS-CoV nucleocapsid protein, the alpha-helical viral protein that interacts with the viral genomic RNA to form the viral nucleocapsid, was reported to begin unfolding at 35°C and was completely denatured at 55°C in phosphate-buffered saline (Wang et al., Citation2004). Since nucleocapsid protein has an RNA-binding domain and is closely associated with the viral genomic RNA, it is possible that disruption of the nucleocapsid protein could leave the viral RNA open to mutagenesis leading to attenuation.
Safety of the GA08/HSp16/08 was demonstrated by giving a 10x dose to 5-day-old broiler chicks. Broiler chicks were used because we wanted to evaluate the characteristics of the vaccine in commercial birds with maternal antibodies. A previous study showed that a high percentage of chicks failed to produce IBV serum antibodies following a single intraocular vaccination at 1 day of age regardless of maternal antibody status (Mondal & Naqi, Citation2001). Our data are consistent with that report since only five out of 25 birds given a single dose of GA08/HSp16/08 produced detectable serum antibodies. It is well known that IgM can be detected soon after an initial IBV infection, and that it wanes quickly (Cavanagh & Gelb Jr, Citation2008). That, plus the fact that the ELISA primarily detects IgG-specific serum antibodies against IBV, could explain the low percentage of antibody-positive chicks following a single vaccination. Our efficacy experiment showed that two doses of the GA08/HSp16/08 induced a protective immune response in maternal antibody-positive birds, which is consistent with previously reported data (Mondal & Naqi, Citation2001). It is not clear whether only one vaccination would be effective.
Sequence analysis and comparison of the parent virus GA08/pass4/08 with the attenuated virus GA08/HSp16/08 showed 41 residue changes (all within the first 282 amino acids) of the 543 residue S1 subunit of the spike gene, which calculates to 92.8% identity, indicating that heat treatment and passage in embryonated eggs did result in genetic changes. However, the pathogenic GA08/08/08 strain passed 16 times in eggs without heat treatment showed 36 amino acid changes in the S1 subunit when compared with the parent GA08/pass4/08 virus, which calculates to 93.9% identity. It is not possible from these data to determine whether the genetic changes were a result of mutations that occurred during virus replication or whether the observed changes were due to selection of existing virus subpopulations in the original inoculum, or both. The S1 gene is the most variable gene within IBV and plays a role in host cell attachment, virus entry, and stimulation of neutralizing and serotype specific antibodies (Cavanagh & Gelb Jr, Citation2008). However, it was recently shown that pathogenicity (attenuation)-related genes are located in the replicase genes (1a/1ab) (Armesto et al., Citation2009). Therefore it is likely that mutations leading to the attenuation of GA08/HSp16/08 are located in the 1a/1ab genes. It logically follows that for an attenuated strain of IBV to induce a neutralizing antibody response, the S1 gene of the attenuated virus ought to be relatively similar to the pathogenic virus. To evaluate the relative similarity of the GA08 viruses, we compared them with each other and with viruses of different serotypes () and found the GA08 viruses including the attenuated heat-treated virus clustered into a distinct group. These data indicated that the GA08/HSp16/08 virus ought to induce neutralizing antibodies against the GA08 virus type, which was verified by the efficacy studies in chickens.
Viruses in an adjacent clade with 80 to 85% similarity were: HN99, a nephropathogenic strain; JAAS/04, a vaccine strain from China; and N1/62, a subgroup I nephropathogenic strain from Australia (Sapats et al., Citation1996; Liu et al., Citation2006). The GA08 virus group also grouped close to the California viruses CA/557/03, CAV/CAV1686/95, CA/CA12495/98 and CAV/CAV9437/95 (78 to 83% similarity), as well as Ark/ArkDPI/81 (77 to 82% similarity), indicating that the spike glycoproteins of these viruses may be related. Although exceptions do occur, typically genetically distant viruses (<89% similarity in S1) that fall into different genetic groups do not cross-protect (Lee et al., Citation2001; Jackwood et al., Citation2007).
It should be acknowledged that although the safety and efficacy testing of the GA08/HSp16/08 virus (pass 16) was performed according to Section 113.327, d, 2, of Title 9 of the Code of Federal Regulations (USDA, Citation1999) for IBV vaccine testing, a rather limited number of birds were used and the dose for safety testing was relatively low. In addition, broilers of commercial origin with potentially undetected maternal immunity to the GA08 virus could have altered the results.
In summary, we were able to attenuate the IBV GA08 type virus, a new variant virus identified in Georgia in 2008, by heat treatment in approximately 10 weeks. This represents an extremely short time compared with 38 to 50 weeks for conventional passage in embryonated eggs. The pathogenic parent virus and attenuated virus had 92.8% amino acid similarity in the S1 glycoprotein and were not genetically similar to other viruses found in the USA. Based on clinical signs, lesions, and challenge virus re-isolation, the attenuated GA08/HSp16/08 virus protected broiler chickens against challenge with the pathogenic GA08 virus.
Acknowledgements
The authors would like to thank Lauren Byrd, Joshua Jackwood, Jamie Phillips, and Sharmi Thor for their help with the sequencing and real-time RT-PCR.
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