Abstract
The S1 genes of isolates of avian coronavirus infectious bronchitis virus (IBV) from commercial chickens in the US and Israel (20 isolates from each country) were studied using reverse transcription-polymerase chain reaction restriction fragment length polymorphism and sequencing. Partial sequences spanning the amino terminus region of S1 from amino acid residues 48 to 219, based on the Beaudette strain, were used for analysis. Phylogenetic clustering and high-sequence identity values were used to identify isolates that appeared to be derived from live IBV vaccines used in the two countries. Novel variant strains, unrelated by S1 sequencing and restriction fragment length polymorphism analyses to reference and vaccine strains, were also identified. Based on S1 sequence identity to available vaccines, the potential to use vaccination to control IBV infections was evaluated. Vaccination with commercial live strains Massachusetts (Mass), Arkansas (Ark) or DE/072/92, generally produced immunity against vaccine-related field isolates displaying high S1 sequence similarities (≥90%) to the respective vaccine strains. Immunization with a bivalent vaccine containing the Mass and Ark strains provided good cross-protection, averaging 81% against challenge with five variant isolates from the US having amino acid identity values ranging from 62 to 69% to Mass and from 68 to 83% to Ark, respectively. In contrast, the H120 vaccine strain induced low levels of protection, ranging from 25 to 58% against variant field isolates from Israel with amino acid identity values from 65 to 67%.
Caractéristiques du gène S1 et efficacité de la vaccination contre les souches de virus de la bronchite infectieuse isolées aux USA et en Israël (1996-2000)
Les gènes S1 des souches de coronavirus aviaire de la bronchite infectieuse (IBV) isolées chez des poulets de chair aux USA et en Israël (20 souches de chaque pays) ont été étudiées en utilisant les techniques de transcription inverse (RT), d'amplification en chaîne par polymérase (PCR), de polymorphisme de taille des fragments de restriction de l'acide nucléique (RFPL) et de séquençage. Les séquences partielles de la région aminée terminale du gène S1 comprise entre les résidus acides aminés 48 et 219, ont été analysées sur la base de la souche Beaudette. Les regroupements phylogénétiques et les valeurs élevées d'identité de séquence ont été utilisés pour identifier les isolats qui apparaissaient être dérivés des vaccins vivants utilisés dans les deux pays. Au vu des résultats du séquençage et des analyses de RFLP, de nouvelles souches variantes ont été identifiées, sans lien avec les souches de référence et les souches vaccinales. En se basant sur l'identité de la séquence de S1 des souches vaccinales disponibles, la possibilité d'utiliser la vaccination pour contrôler les infections à IBV a été évaluée. La vaccination avec des souches vivantes commerciales Massachusetts (Mass), Arkansas (Ark) ou DE/072/92, a généralement entraîné une immunité vis-à-vis des souches du terrain ayant un lien avec les souches vaccinales montrant des similarités élevées de la séquence de S1 (≥90%) avec celles des souches vaccinales respectives. L'immunisation avec un vaccin bivalent contenant les souches Mass et Ark a conféré une bonne protection croisée, de 81% en moyenne, vis-à-vis d’épreuves avec cinq virus variants isolés aux USA ayant des niveaux d'identité en acides aminés allant de 62 à 69% vis-à-vis de la souche Mass et de 68 à 83% vis-à-vis de la souche Ark. Par contre, la souche vaccinale H120 a induit des taux faibles de protection, allant de 25 à 58% vis-à-vis des variants isolés en Israël ayant des niveaux d'identité en acides aminés allant de 65 à 67%.
Charakteristika des S1-Gens und Wirksamkeit der Vakzination gegen Feldisolate des Virus der infektiösen Bronchitis aus den USA und Israel (1996–2000)
Die S1-Gene von Isolaten des aviären Coronavirus infektiöse Bronchitisvirus (IBV) aus kommerziellen Hühnern in den USA und Israel (20 Isolate aus jedem Land) wurden mittels reverser Transkriptions (RT)-Polymerasekettenreaktion (PCR), Restriktionsfragmentlängen-polymorphismus (RFLP) und Sequenzierung untersucht. Sequenzstücke, die basierend auf dem Beaudette-Stamm, die Aminoendregion des S1 mit den Aminosäurenresiduen 48-219 umfassten, wurden für die Analyse verwendet. Phylogenetische Clusterung und hohe Sequenzübereinstimmungswerte dienten dazu, die Isolate zu identifizieren, die wahrscheinlich von in den beiden Ländern verwendeten Lebendvirusvakzinen abstammten. Neue Variantstämme, die aufgrund der S1-Sequenzierung und der RFLP-Analyse nicht mit den Referenz- und Vakzinestämmen verwandt waren, wurden auch nachgewiesen. Basierend auf der S1-Sequenzübereinstimmung der untersuchten Isolate mit den verfügbaren Vakzinen wurde die Eignung der Vakzination zur Bekämpfung von IBV-Infektionen ermittelt. Die Vakzination mit den kommerziellen Lebendvirusstämmen Massachusetts (Mass), Arkansas (Ark) oder DE/072/92 erzeugte im allgemeinen eine Immunität gegen Vakzine-verwandte Feldisolate mit hohen S1-Sequenzähnlichkeiten (≥90%) zu den entsprechenden Vakzinestämmen. Die Immunisierung mit einer bivalenten Vakzine mit den Stämmen Mass und Ark erzeugte einen guten Kreuzschutz von im Mittel 81% gegen Belastungsinfektionen mit fünf Variantstämmen aus den USA, die in ihren Aminosäurensequenzen zu 62–69% mit Mass bzw. zu 68-83% mit Ark übereinstimmten. Im Gegensatz dazu induzierte der Vakzinestamm H120 nur eine geringe Schutzwirkung von 25–58% gegen Variant-Feldisolate aus Israel, die zum Impfstamm Übereinstimmungswerte der Aminosäuren von 65–67% aufwiesen.
Características del gen S1 y eficacia de la vacunación frente a aislamientos de campo del virus de la bronquitis infecciosa de Estados Unidos e Israel (1996–2000)
Se estudió el gen S1 de aislamientos del coronavirus de la bronquitis infecciosa aviar (IBV) procedentes de pollos comerciales de Estados Unidos e Israel (20 aislamientos de cada país) mediante el uso de la transcripción reversa (RT), reacción en cadena de la polimerasa (PCR), polimorfismo de la longitud de los fragmentos de restricción y secuenciación. Se usaron para el análisis, secuencias parciales que abarcaban la región amino terminal de la S1 entre los residuos aminoacídicos 48–219, en base a la cepa Beaudette. El agrupamiento filogenético y los valores de mayor identidad de secuencias se usaron para identificar aislados que parecían proceder de vacunas vivas de IBV utilizadas en ambos países. También se identificaron nuevas cepas variantes, no relacionadas mediante secuenciación de la S1 y análisis de RLFP a las cepas vacunales y de referencia. Se evaluó la posibilidad de usar la vacunación en el control de las infecciones por IBV, basándose en la identidad de las secuencias de la S1 respecto las vacunas disponibles. La vacunación con cepas vivas comerciales Massachussets (Mass), Arkansas (Ark) o DE/072/92, generalmente produjo inmunidad frente a los aislamientos de campo relacionados con las vacunas, mostrando elevadas similitudes (≥90%) de la secuencia de la S1 con las respectivas cepas vacunales. La inmunización con una vacuna bivalente que contiene las cepas Mass y Ark proporcionó buena protección cruzada con una media del 81% frente a la infección experimental con cinco aislamientos variantes de US con una identidad aminoacídica entre 62–69% frente a Mass y 68–83% frente a Ark, respectivamente. Por el contrario, la cepa vacunal H120 indujo niveles bajos de protección, oscilando entre el 25–58% frente a los aislamientos variantes de campo de Israel con identidades aminoacídicas entre el 65–67%.
Introduction
Infectious bronchitis (IB) is among the most common and difficult of all poultry diseases to control. IB is highly contagious and results in significant economic losses in commercial broiler, layer and breeder chickens. The causative coronavirus, infectious bronchitis virus (IBV), frequently causes respiratory disease in young chickens and egg production losses in hens. In addition, some strains of the virus exhibit a renal (kidney) tropism and produce up to 30% mortality in affected flocks.
IBV readily undergoes mutation in chickens resulting in the emergence of new variant serotypes and genotypes. Mutations occur frequently in hypervariable regions of the S1 subunit of the envelope spike (S) glycoprotein gene. The S1 protein is responsible for infection of the host cell (Cavanagh & Davis, Citation1986; Koch et al., Citation1990; Ignjatovic & Galli, Citation1994), inducing virus-neutralizing antibody (Cavanagh et al., Citation1988) and immunity (Cavanagh et al., Citation1997). New variant strains may differ as much as 55% in their S1 amino acid sequence compared with vaccine strains (Kusters et al., Citation1989; Gelb et al., Citation1997). Many “new” variants have been isolated from commercial flocks (Davelaar et al., Citation1984; Kusters et al., Citation1989; Gelb et al., Citation1991, Citation1997, Citation2001; Cavanagh et al., Citation1992; Gough et al., Citation1992, Citation1996; Capua et al., Citation1994; Moore et al., Citation1998; Fabio et al., Citation2000; Lee and Jackwood, Citation2001; Zeigler et al., Citation2002; Schikora et al., Citation2003; Meir et al., Citation2004). Two genetic-based strategies have been used in the US and Israel to identify field strains; restriction fragment length polymorphism (RFLP) analysis (Kwon et al., Citation1993; Meir et al., Citation2004), and reverse transcription-polymerase chain reaction (RT-PCR) product cycle sequencing (Kingham et al., Citation2000).
The purpose of this research was to characterize 40 IBV isolates from diseased flocks in the US and Israel by partial sequencing of the S1 protein. Vaccine strains with the highest sequence identity to the field isolates were evaluated for their ability to protect chickens in challenge of immunity trials.
Materials and Methods
Viruses
Twenty IBV field isolates from the US () and an equal number from Israel () were recovered from commercial meat-type (broiler) and egg-type (layer) chicken flocks between 1996 and 2000. The isolates as well as reference strains for Arkansas (Ark) DPI, Connecticut (Conn), DE/072/92 (Delaware 072), JMK, and Massachusetts (Mass) 41 are maintained as allantoic fluid seed stocks in the authors’ laboratories.
Table 1. IBV field isolates from the US
Table 2. IBV field isolates from Israel
Commercially available live IBV vaccines currently licensed for use in the US and Israel were used to evaluate their efficacy against challenge with selected field isolates recovered from the respective countries. Vaccines containing serotypes from the US were Mass, Ark, and DE/072/92. A commercial H120 strain vaccine from Israel was used. The S1 genes of the vaccines and reference strains were characterized by RFLP and sequencing to provide a basis of comparison with the field isolates.
Field isolates and vaccines used for S1 RFLP and sequencing were inactivated by treating approximately 2 ml allantoic fluids or rehydrated lyophilized vaccines with an equal volume of molecular biology-grade phenol (pH 4.3) (Fisher Scientific, Fairlawn, New Jersey, USA). Following inactivation, field isolates and vaccines from both countries were exchanged for S1 gene analyses. Sequencing was performed in the US and RFLP analysis was performed in Israel.
S1 gene RFLP analysis
IBV RNA was extracted using a kit (QIAamp Viral RNA Mini Kit; Qiagen, Inc., Valencia, California, USA). RT-PCR RFLP was performed to amplify the complete S1 gene using forward primer S1 OLIGO5′ and reverse primer S1 OLIGO3′, yielding an amplicon of approximately 1700 nucleotides (Kwon et al., Citation1993; Meir et al., Citation2004). PCR products were digested with three commonly used restriction enzymes: BstYI, HaeIII, and XcmI (Kwon et al., Citation1993). Digested PCR products were separated by electrophoresis in 2% agarose gels stained with ethidium bromide. Restriction patterns of the field isolates were compared with patterns observed with digested vaccine and reference strain PCR products.
S1 gene PCR product cycle sequencing
Viral RNA was extracted (QIAamp Viral RNA Mini Kit; Qiagen, Inc.) from allantoic fluid stocks of the field isolates or directly from the vaccines. RNA was eluted in sterile diethyl pyrocarbonate-treated water and stored at −70°C until used for RT-PCR.
RT was performed on viral RNA using the GeneAmp RNA PCR Core Kit (Applied Biosystems, Foster City, California, USA). Approximately 2 μl extracted viral RNA was used to synthesize cDNA. Amplification of the 5′ end of the S1 gene was performed using the forward primer S1 OLIGO5′ (Kwon et al., Citation1993) and the reverse primer CK2 (Keeler et al., Citation1998), resulting in an amplicon of approximately 700 nucleotides. PCR was performed as described previously (Kingham et al., Citation2000) with the exception that extension was performed at 60°C. PCR products were cut from 1.8% agarose gels, purified with the QIAquick Gel Extraction Kit (Qiagen, Inc.) and DNA was quantified as described (Kingham et al., Citation2000).
Purified RT-PCR products were sequenced in the forward direction using primer S1 OLIGO5′ and in the reverse direction using primer CK2 (Kingham et al., Citation2000). Analysis was performed using partial sequences spanning the amino terminus region of S1 from amino acid residue 48 to 219, based on the Beaudette strain (GenBank accession number NP 04083). The gene fragment encompasses hypervariable region 1 and the majority of hypervariable region 2 (Cavanagh et al., Citation1988; Kusters et al., Citation1989). Analysis of the partial S1 sequences obtained by our laboratory and from GenBank and EMBL databases was performed using the CLUSTAL V (Higgins & Sharp, Citation1988) package of DNAStar (MegAlign, Version 1.03, 1993; DNAStar, Inc., Madison, Wisconsin, USA).
Comparison of partial and complete S1 sequences of highly related strains will yield virtually identical S1 amino acid identity values. In the case of variant strains that differ extensively in their S1 amino acid sequences, the identity values for the partial sequences will be on average, approximately up to 9% lower than for the complete S1 sequences. The lower identity of isolates using analyses of the partial sequences is a reflection of the higher relative variability associated with the 5′ portion of the gene compared with that of the full-length S1 gene.
Vaccination-challenge of immunity trials
Three vaccination-challenge of immunity trials were performed. Trial 1 evaluated the efficacy of commercial live IBV vaccines containing the Ark DPI, DE/072/92, or Mass strains versus challenge with US field isolates displaying a high degree of S1 identity (≥91%) to the vaccines. Trial 2 examined the efficacy of a commercial vaccine containing the Mass and Ark strains versus challenge with variant field isolates CA/510/99, PA/171/99, PA/5083/99, PA/5344/98, and MN/113575/99 from the US. Trial 3 assessed the efficacy of a commercial H120 strain vaccine versus challenge against field isolates IS/64714/96, IS/385/97, IS/585/98, IS/665/98, and IS/720/99 from Israel.
The procedures for each of the three vaccination-challenge trials were as follows. Two-week-old specific pathogen free (SPF) Leghorn chickens were obtained (Charles River SPAFAS, North Franklin, Connecticut, USA) and assigned to groups of 10 to 13 birds per group. The chickens were vaccinated via eye drop with recommended doses of commercial modified live IBV vaccines and were placed in Horsfall-type isolation units. Four weeks post vaccination, additional SPF leghorns were obtained from the supplier and five birds were housed with each of the IBV challenge virus treatments as challenge controls. Vaccinated and challenge control chickens were inoculated by eye drop with approximately 104.0 to 104.5 EID50 per chick of the field isolates. Five days after challenge, tracheal swabbing was performed on all chickens and were used for virus re-isolation attempts (Ladman et al., Citation2002). Virus isolations were performed by a single passage in 9-day-old to 11-day-old SPF embryonated eggs via chorioallantoic sac inoculation. Percentage protection values for each treatment group were calculated to determine the level of immunity provided by vaccination against challenge with the specific field isolates.
Results
RFLP and S1 gene sequence analyses
US Isolates
IBV field isolates from the US were placed into 15 groups based on RFLP analysis using restriction enzymes, BstYI, HaeIII, and XcmI (). Six groups (A-1, A-2, A-3, B, C-1 and C-2) consisted of 11 isolates that were identified as various reference strains with identical or similar RFLP patterns. Field isolates in Group A-1 (AL/2975/00, AL/3701/00) were identical by RFLP to the DE/072/92 strain. Isolates in Groups A-2 (DE/322/99, DE/406/99, GA/5425/99) and A-3 (PA/5173/98) displayed similar but not identical RFLP patterns to the DE/072/92 genotype and were identified as DE/072/92-like. HaeIII restriction produced an additional fragment in Group A-2 (750 nucleotides) and Group A-3 (650 nucleotides). Group B isolates PA/5083/99 and PA/4327/97 produced RFLP patterns consistent with reference strain PA/Wolgemuth/98. Group C-1 consisted of a recent field isolate PA/171/99 considered now to be a reference strain (Zeigler et al., Citation2002). RFLP analysis of PA/171/99 and MD/106/00 (Group C-2) yielded identical BstYI and HaeIII polymorphisms but a different XcmI pattern.
Table 3. RFLP results of IBV field isolates from the US
Isolates in six of the 15 RFLP groups could not be identified as known S1 genotypes because they were novel variants unrelated to known reference strains (). Variant IBV isolates were placed into six RFLP groups (E, F-1, F-2, H, J, and K) each containing a single isolate. All of the novel variant strains displayed unique RFLP patterns. Moreover, it was noted that isolates in Group E (CA/1249/99), Group J (MN/11375/99), and Group K (PA/1220/98) displayed S1 sequence identity values to reference strains (≤75%) that were consistent with previous cut-offs for identifying a strain as a novel variant (Kingham et al., Citation2000). Interestingly, novel variant isolates in Group F-1 (CA/510/99), Group F-2 (CA/3402/99), and Group H (PA/5344/98) had S1 sequence identity values to reference strains that ranged from 83 to 85%.
Finally, field isolates in Groups D, G, and I also did not display RFLP patterns consistent with vaccine or reference strain genotypes (). However, isolates in these groups had high S1 sequence identity values with vaccine or reference strain genotypes. Identity values of the isolates in groups D (MD/183/99) and G (MD/30/99) were ≥98% to reference strain Ark/1529/95 (Nix et al., Citation2000). Isolate DE/51/99 of Group I had RFLP patterns similar to the JMK strain, but its sequence was consistent (≥94% similar) with the Mass 41 genotype.
A phylogeny of the partial S1 amino acid sequences of US field isolates, commercial vaccines and selected reference strains is shown in . The respective reference and vaccine strains for Ark, Conn, DE/072/92, and Mass genotypes clustered together. S1 amino acid sequences of commercial vaccines and their respective reference strains were highly related, ranging from 98 to 100% identity (). The field isolates demonstrated extensive sequence variability to the reference and vaccine strains. PA/1220/98, CA/1249/99, and MN/11375/99 were highly novel variants and were distantly related to other US isolates, reference strains and vaccines. Isolates MD/30/99 and MD/183/99 were found to be highly similar (≥97%) to Ark reference strain Ark/1529/95. PA/4327/97 and PA/5083/99 were ≥95% similar to PA/Wolgemuth/98. PA/171/99 and MD/106/00 were 98% similar to each other. DE/51/99 was highly similar (94%) to Mass 41 and even more closely related (98 to 99%) to the Mass vaccines. Isolates AL/2975/00, AL/3701/00, DE/322/99, DE/406/99, GA/5425/99 and PA/5173/98 were related to DE/072/92, with identity values ranging from 89 to 92%.
Figure 1. Phylogenetic tree showing partial (Beaudette GenBank accession number NP 04083 amino acid position 48 [nucleotide 144] to 219 [nucleotide 657]) S1 gene inter-relationships between IBV field isolates from the US, commercial vaccines and reference (ref) strains for Arkansas, Connecticut, DE/072/92, Massachusetts and JMK (reference strain only). * Vaccines used for immunization in the vaccination-challenge of immunity trials ( and ).
![Figure 1. Phylogenetic tree showing partial (Beaudette GenBank accession number NP 04083 amino acid position 48 [nucleotide 144] to 219 [nucleotide 657]) S1 gene inter-relationships between IBV field isolates from the US, commercial vaccines and reference (ref) strains for Arkansas, Connecticut, DE/072/92, Massachusetts and JMK (reference strain only). * Vaccines used for immunization in the vaccination-challenge of immunity trials (Tables 5 and 6).](/cms/asset/ce284d21-a4b8-4652-9068-b64f57085255/cavp_a_0194_o_fig001.gif)
Israeli isolates
IBV field isolates from Israel were placed into four RFLP groups (). Group A contained 10 of 20 Israeli isolates (IS/236/96, IS/287/96, IS/361/97, IS/373/97, IS/385/97, IS/388/97, IS/415/97, IS/617/98, IS/665/98, and IS/51355/98) that had RFLP patterns consistent with the Mass genotype. Group B isolates (IS/188/96, IS/222/96, IS/251/96, and IS/64714/96) were identified as Variant 1, and Group C viruses (IS/223/96, IS/378/97, IS/572/98, IS/585/98, and IS/589/98) were found to be Variant 2 by RFLP. One isolate, IS/720/99 (Group D) had a unique RFLP pattern and a low sequence identity to reference strains, and was therefore designated a novel variant.
Table 4. RFLP results of IBV field isolates from Israel
S1 analysis using the partial amino acid sequences of the Israeli field isolates supported the RFLP findings (). S1 sequence identity values of the isolates identified by RFLP as Mass ranged from 95 to 100% and were highly related (95 to 100%) to the three commercial H120 vaccines. Isolates representing the Variant 1 (Group B) and Variant 2 (Group C) viruses were highly related to their respective genotypic groups, displaying S1 sequence identity values ranging from 95 to 100% and from 95 to 99%, respectively.
The S1 phylogeny of the Israeli field isolates, commercial vaccines and selected reference strains are shown in . Nineteen of 20 isolates clustered into three clades representing Mass, Variant 1, and Variant 2 genotypes. Isolate IS/720/99, represented a new genotype.
Figure 2. Phylogenetic tree showing partial (Beaudette GenBank accession number NP 04083 amino acid position 48 [nucleotide 144] to 219 [nucleotide 657]) S1 gene inter-relationships between IBV field isolates from Israel, selected reference strains and commercial H120 vaccines. * H120 Vaccine 1 used for immunization in the vaccination-challenge of immunity trial ().
![Figure 2. Phylogenetic tree showing partial (Beaudette GenBank accession number NP 04083 amino acid position 48 [nucleotide 144] to 219 [nucleotide 657]) S1 gene inter-relationships between IBV field isolates from Israel, selected reference strains and commercial H120 vaccines. * H120 Vaccine 1 used for immunization in the vaccination-challenge of immunity trial (Table 7).](/cms/asset/e326b96d-31c8-4770-97c2-4bb21d36ef65/cavp_a_0194_o_fig002.gif)
Vaccination-challenge of immunity trials
Trial 1: Efficacy of commercial vaccines against challenge with US field isolates with a high degree of S1 identity >91%) to the vaccines
Vaccination with commercial IBV live strains generally conferred solid immunity against field isolates that shared a high S1 sequence identity with the field isolate used for challenge (). Vaccination with DE/072/92 resulted in partial protection (55%) against challenge with isolate DE/406/99. The isolates had S1 sequence identity values ranging from 91 to 99% to the vaccine strains used in the trial.
Table 5. Protection of chickens following vaccination with IBV strains and challenged with US field isolates with high (>91%) S1 sequence (amino acid) similarity values to the vaccines
Trial 2: Efficacy of a commercial vaccine containing the Mass and Ark strains against challenge with variant field isolates CA/510/99, PA/171/99, PA/5083/99, PA/5344/98, and MN/113575/99 from the US
Mass and Ark strain combination vaccination produced good levels (≥70% protection) of immunity against challenge with variant field isolates CA/510/99, PA/171/99, PA/5083/99, and MN/113575/99 (). Vaccination with Mass and Ark produced partial protection (50%) chickens following challenge with isolate PA/5344/98. The isolates had S1 sequence identity values ranging from 62 to 69% to the Mass vaccine strain and from 68 to 83% to the Ark vaccine strain used in the trial. Only three of five (60%) of the susceptible challenge control chickens were positive for IBV following challenge with PA/5083/99, suggesting that the dose given to the controls as well as the Mass and Ark vaccinates for this treatment was low. It is possible that the level of protection (91%) in the Mass and Ark vaccinates actually may be lower than the reported value.
Table 6. Protection of chickens following vaccination with strains Massachusetts and Arkansas against challenge with variant US field isolates
Trial 3: Efficacy of a commercial vaccine containing the H120 strain against challenge with field isolates from Israel (IS/64714/96 [Variant 1], IS/385/97, IS/585/98 [Variant 2], IS/665/98, and IS/720/99)
Chickens vaccinated with H120 were well protected (83%) from challenge with IS/665/98, an isolate highly related to the vaccine. However, H120 protection versus challenge with IS/385/97, also highly related (96%) to H120, resulted in only partial (58%) protection. Poor protection (25 to 58%) of H120-vaccinated chickens was noted versus challenge with variant isolates IS/64714/96 (Variant 1), IS/585/98 (Variant 2) and IS/720/99. The three Israeli variant isolates used in the trial had S1 sequence identity values of 65 to 67% to the H120 vaccine strain.
Discussion
S1 partial gene sequencing and RFLP analyses were used to determine the relationship of field isolates to vaccine strains, previously described reference strains or new variant genotypes. Field isolates from the US and Israel could be placed into four categories: isolates highly related to vaccines; isolates highly related to reference strains; isolates related to, yet distinguishable from, vaccines or reference strains; or variants unrelated to vaccines or reference strains.
Nine of 40 isolates in the study were highly related (>99%) by sequencing to Mass serotypes vaccines. US isolate DE/51/99 was most highly related to Mass vaccines used in the US (). Eight Israeli isolates (IS/236/96, IS/361/97, IS/373/97, IS/388/97, IS/415/97, IS/617/98, IS/665/98, and IS/51355/98) were highly related to H120 vaccines used in Israel ( and ). IS/361/97 and IS/388/97 were isolated from broilers with renal lesions consistent with nephropathogenic IB. H120 vaccine may have persisted in the birds and was inadvertently recovered from the flock, but was not the cause of the renal disease. A pathogenesis study could establish the potential role of the isolates in the outbreak.
Isolates highly related to reference strains included IS/222/96, IS/251/96, and IS/64714/96 to Variant 1 (≥98% S1 identity) and IS/223/96, IS/572/98, IS/585/98, and IS/589/98 to Variant 2 (99% S1 identity). Variant 1 and Variant 2 viruses were first reported in Israel in 2001 (Callison et al., Citation2001). Variant 1 viruses are members of the genotype that includes strains 4/91 and 7/93B from the UK (Gough et al., Citation1992; Parsons et al., Citation1992).
US isolates related to, yet distinguishable from, vaccines or reference strains were also identified ( and ). MD/30/99 and MD/183/99 were related to, yet distinguishable from, Ark DPI vaccine with S1 similarities of 94 and 93%, respectively. The isolates had the highest S1 sequence identity (98%) to reference strain Ark/1529/95, recovered from broilers from the Delmarva Peninsula region in 1995 ( and ). Ark/1529/95 was identified as a quasi-species in Ark DPI vaccines, and was thought to have reverted to virulence through back-passage (Nix et al., Citation2000).
Isolates AL/2975/00, AL/3701/00, DE/322/99, DE/406/99, GA/5425/99 and PA/5173/98 from the Delmarva and Southeast regions of the US were also related to, yet distinguishable from, the DE/072/92 reference strain (S1 identity 89 to 92%) and the commercial vaccine (S1 identity 91 to 92%). The relatively low identity values to the vaccine suggest the field isolates originated from an unknown source in the field and were not derived from either the DE/072/92 reference strain or the commercial vaccine. Futhermore, the Delmarva and Southeast isolates were also different from each other with S1 similarities from 89 to 92% (data not shown). The identification of three related yet distinguishable DE/072/92 RFLP groups (A-1, A-2 and A-3) also suggested that the field isolates are similar but not identical. Our findings support initial (Gelb et al., Citation1997) and later reports (Lee et al., Citation2001) demonstrating the diversity within the DE/072/92 genotype. It appears that DE/072/92 genotypes are evolving from multiple origins in the field.
Among Israeli isolates, two were related to, yet distinguishable from, the H120 vaccine by S1 sequence and RFLP analyses ( and ). Mass genotypes IS/287/96 and IS/385/97 were not highly related to H120 (95 and 96%, respectively), but were also not highly related to Mass 41 (92 and 94% identity, respectively). The isolates may represent indigenous Mass genotype strains circulating in Israeli poultry.
US isolates PA/4327/97 and PA/5083/99 were related to, yet distinguishable from, reference strain PA/Wolgemuth/98, one of two unique genotypes found during a nephropathogenic outbreak in Pennsylvania (Ziegler et al., Citation2002). PA/4327/97 and PA/5083/99 had sequence identity values of 94 and 96%, respectively, compared with PA/Wolgemuth/98. S1 similarities of PA/4327/97, PA/5083/99, and PA/Wolgemuth/98 to the other genotype identified in the outbreak, PA/171/99, ranged from only 80 to 85% (Ziegler et al., Citation2002). RFLP analysis supported the sequence findings indicating the PA/Wolgemuth/98 and PA/171/99 genotypes are different ().
US isolates CA/1249/99, MN/11375/99, and PA/1220/98 had unique S1 sequences that classified them as variants unrelated to vaccines or reference strains ( and ). Isolates CA/1249/99, MN/11375/99, and PA/1220/98 had low identity values to known genotypes. RFLP analysis also identified these isolates as having unique restriction patterns.
Isolate IS/720/99 was also classified as a variant ( and ). In Israel, isolates of the same genotype continue to cause IB but recent cases have been associated with renal disease (Meir et al., Citation2004). In 2001, we identified another highly related isolate of the IS/720/99 genotype from Egypt (Egypt/Beni-Seuf/01; AF395531) (Abdel-Moneim et al., Citation2002), associated with both renal and respiratory forms of the disease.
Three US isolates could not be classified as either related to a vaccine or reference strain or a variant by sequencing. S1 similarities for CA/510/99 (83% to Ark DPI), CA/3402/99 (83% to Ark DPI), and PA/5344/98 (85% to JMK) were higher than those typically associated with variants (). Until the year 2000, we considered partial S1 sequences of variant genotypes to be ≤75% similar to a reference or vaccine strain (Kingham et al., Citation2000). Since that time, we have encountered several isolates such as CA/510/99, CA/3402/99, and PA/5344/98 with S1 identity values up to 85%. Antigenic characterization using virus neutralization and reciprocal cross-challenge studies in chickens may provide biological relevance to “intermediate” S1 genetic inter-relationships.
In challenge of immunity trials, IBV vaccines with high S1 identity values (≥93%) were protective using four of six field isolates ( and ). Other reports (Cavanagh et al., Citation1997; Gelb et al., Citation1997) using non-attenuated field strains for immunization have described similar results. Partial protection was observed against the remaining two field isolates. DE/072/92 immunization versus DE/406/99 challenge produced 55% protection, and H120 vaccination resulted in 58% protection, against IS/385/97. Hodgson et al. (Citation2004) reported that a recombinant Beaudette with the S gene of Mass 41 induced better protection against challenge with Mass 41 than the original non-recombinant Beaudette strain even though the S1 proteins differed by only 5%. Factors such as differences in epitopes of the vaccine strains and challenge viruses used may have influenced the challenge findings. Moreover, protection results from challenge assays sometimes display a degree of variability that is a function of the relatively few chickens used.
Table 7. Relationship of protection and S1 sequence (amino acid) similarity values of IS field isolates and a commercial live IBV vaccine containing the H120 strain
The degree of in vivo cross-protection often declines with decreasing S1 sequence homology between isolates used for IBV immunization and challenge (Cavanagh et al., Citation1997; Gelb et al., Citation1997). H120 vaccination provided little protection versus challenge with IS/720/99 (36%) and Variant 2 (25%). This finding is of concern since H120 is the only licensed live vaccine strain in Israel.
In contrast, immunization with a heterologous vaccine strain(s) may cross-protect even when there are considerable differences in S1 similarities between the vaccine and challenge strains (). The phenomenon of IBV cross-protection has been recognized for many years (Hofstad, Citation1981; Cook et al., Citation1999) and has been attributed to the immune response to shared S1 as well as other IBV gene epitopes (Cavanagh et al., Citation1997). Immunization with a bivalent vaccine containing the Mass and Ark strains provided cross-protection against challenge with IBV field strains (Gelb et al., Citation1991). Protection X¯=81%, value not in ) was observed against challenge with CA/510/99, PA/171/99, PA/5083/99, PA/5344/98, and MN/113575/99. H120 vaccination provided partial protection (58%) against Variant 1 (). The findings illustrate the importance of evaluating the efficacy of vaccination against isolates that may be quite unique based on sequencing.
Discrepancies between RFLP and sequencing results occurred with US isolates DE/51/99, MD/183/99 and MD/30/99 (). DE/51/99 was identified as JMK by RFLP and as Mass by sequencing. RE analysis of available S1 sequences for DE/51/99 indicated that the discrepancy could be explained by a point mutation(s) at BstYI and XcmI restriction sites, which yielded two fragments instead of three. Sequencing findings identifying DE/51/99 as a Mass genotype were supported by challenge of immunity results (). MD/183/99 and MD/30/99 were identified as different genotypes (RFLP Group D and Group G, respectively). However, both isolates were highly related (98%) by sequencing to Ark/1529/95 (). RE analysis using available partial sequences for MD/183/99 and MD/30/99 were not helpful in resolving the discrepancy. Challenge of immunity results supported the sequencing findings that MD/183/99 and MD/30/99 were Ark genotypes ().
S1 sequence and RFLP analyses can be used to identify field strains related to vaccines and novel variants. Comparison and analysis of sequences of unknown filed isolates with reference strains for establishing potential relatedness is an advantage of sequencing versus RFLP.
Although S1 gene-based diagnostics have made IBV identification faster, the challenge of disease control remains. Controlling IB is quite feasible when the causative field strain and an available vaccine strain share a high degree of sequence identity. However, many field isolates have low (≤85%) S1 identity values or unique RFLP patterns compared with vaccines. In these situations, the options for controlling the disease may be limited. Selection of monovalent or bivalent vaccines known to induce heterologous cross-protection could yield improved performance. When possible it is important to periodically evaluate the cross-protective capabilities of vaccines versus recently recovered field isolates. Outcomes of these studies will provide valuable information on the practical use of existing vaccines and the potential need for new ones.
Translations of the abstract in French, German and Spanish are available on the Avian Pathology website.
Acknowledgments
This research was supported by Research Grant No. US-3068-98 from BARD, the United States–Israel Binational Agricultural Research and Development Fund.
Related Research Data
References
- Abdel-Moneim AS Madbouly HM Gelb J Jr Ladman BS (2002) Isolation and identification of Egypt/Beni-Seuf /01 a novel genotype of infectious bronchitis virus Veterinary Medical Journal Giza 50 1065 1078
- Callison , SA , Jackwood , MW and Hilt , DA . (2001) . Molecular characterization of infectious bronchitis virus isolates foreign to the United States and comparison with United States isolates . Avian Diseases , 45 : 492 – 499 .
- Capua , I , Gough , RE , Mancini , M , Casaccia , C and Weiss , CA . (1994) . A “novel” infectious bronchitis strain infecting broiler chickens in Italy . Journal of Veterinary Medicine Series B , 41 : 83 – 89 .
- Cavanagh , D and Davis , PJ . (1986) . Coronavirus IBV: removal of spike glycoprotein S1 by urea abolishes infectivity and hemagglutination but not attachment to cells . Journal of General Virology , 67 : 1443 – 1453 .
- Cavanagh , D , Davis , PJ and Mockett , AP . (1988) . Amino acids within hypervariable region 1 of avian coronavirus IBV (Massachusetts serotype) spike glycoprotein are associated with neutralization epitopes . Virus Research , 11 : 141 – 149 .
- Cavanagh , D , Davis , PJ , Cook , JKA , Li , D , Kant , A and Koch , G . (1992) . Location of the amino acid differences in the S1 spike glycoprotein subunit of closely related serotypes of infectious bronchitis virus . Avian Pathology , 21 : 33 – 43 .
- Cavanagh , D , Ellis , MM and Cook , JKA . (1997) . Relationship between sequence variation in the S1 spike protein of infectious bronchitis virus and the extent of cross-protection in vivo . Avian Pathology , 26 : 63 – 74 .
- Cook , JKA , Orbell , SJ , Woods , MA and Huggins , MB . (1999) . Breadth of protection of the respiratory tract by different live-attenuated infectious bronchitis vaccines against challenge with infectious bronchitis viruses of heterologous serotypes . Avian Pathology , 28 : 477 – 485 .
- Davelaar , FG , Kouwenhoven , B and Burger , AG . (1984) . Occurrence and significance of infectious bronchitis virus variant strains in egg and broiler production in the Netherlands . Veterinary Quaterly , 6 : 114 – 120 .
- Fabio , Jdi , Rossini , LI , Orbell , SJ , Paul , G , Huggins , MB , Malo , A , Silva , BGM and Cook , JKA . (2000) . Characterization of infectious bronchitis viruses isolated from outbreaks of disease in commercial flocks in Brazil . Avian Diseases , 44 : 582 – 589 .
- Gelb J Jr Wolff JB Moran CA (1991) Variant serotypes of infectious bronchitis virus isolated from commercial layer and broiler chickens Avian Diseases 35 82 87
- Gelb J Jr Keeler CL Nix WA Rosenberger JK Cloud SS (1997) Antigenic and S-1 genomic characterization of the Delaware variant serotype of infectious bronchitis virus Avian Diseases 41 661 669
- Gelb J Jr Ladman BS Tamayo M Gonzalez M Sivanandan V (2001) Novel infectious bronchitis virus S1 genotypes in Mexico 1998–1999 Avian Diseases 45 1060 1063
- Gough , RE , Randall , CJ , Dagless , M , Alexander , DJ , Cox , WJ and Pearson , D . (1992) . A “new” strain of infectious bronchitis virus infecting domestic fowl in Great Britain . Veterinary Record , 130 : 493 – 494 .
- Gough , RE , Cox , WJ , Gutierrez , E , MacKenzie , G , Wood , AM and Dagless , MD . (1996) . Isolation of “variant” strains of infectious bronchitis virus from vaccinated chickens in Great Britain . Veterinary Record , 139 : 552
- Higgins , DG and Sharp , PM . (1988) . CLUSTAL: a package for performing multiple sequence alignment on a microcomputer . Gene , 73 : 237 – 244 .
- Hodgson , T , Casais , R , Dove , B , Britton , P and Cavanagh , D . (2004) . Recombinant infectious bronchitis coronavirus Beaudette with the spike protein gene of the pathogenic M41 strain remains attenuated but induces protective immunity . Journal of Virology , 78 : 13804 – 13811 .
- Hofstad , MS . (1981) . Cross-immunity in chickens using seven isolates of avian infectious bronchitis virus . Avian Diseases , 25 : 650 – 654 .
- Ignjatovic , J and Galli , L . (1994) . The S1 glycoprotein but not the N or M proteins of avian infectious bronchitis virus induces protection in vaccinated chickens . Archives of Virology , 138 : 117 – 134 .
- Keeler CL Jr Reed KL Nix WA Gelb J Jr (1998) Serotype identification of avian infectious bronchitis virus by RT-PCR of the peplomer (S-1) gene Avian Diseases 42 275 284
- Kingham BF Keeler CL Jr Nix WA Ladman BS Gelb J Jr (2000) Identification of avian infectious bronchitis virus by direct automated cycle sequencing of the S1 Gene Avian Diseases 44 325 335
- Koch , G , Hartog , L , Kant , A and van Roozelaar , DJ . (1990) . Antigenic domains of the peplomer protein of avian infectious bronchitis virus: correlation with biological functions . Journal of General Virology , 71 : 1929 – 1935 .
- Kusters , JG , Niesters , HG , Lenstra , JA , Horzinek , MC and van der Zeijst , BA . (1989) . Phylogeny of antigenic variants of avian coronavirus IBV . Virology , 169 : 217 – 221 .
- Kwon HM Jackwood MW Gelb J Jr (1993) Differentiation of infectious bronchitis virus serotypes using the polymerase chain reaction and restriction fragment length polymorphism analysis Avian Diseases 37 194 202
- Ladman BS Pope CR Ziegler AF Swieczkowski T Callahan JM Gelb J Jr (2002) Protection of chickens following live and inactivated virus vaccination against challenge with nephropathogenic infectious bronchitis virus PA/Wolgemuth/98 Avian Diseases 46 938 944
- Lee , CW and Jackwood , MW . (2001) . Origin and evolution of the Georgia 98 (GA98), a new serotype of infectious bronchitis virus . Virus Research , 80 : 33 – 39 .
- Lee , CW , Hilt , DA and Jackwood , MW . (2001) . Identification and analysis of the Georgia 98 serotype of infectious bronchitis virus . Avian Diseases , 45 : 164 – 172 .
- Meir , R , Rosenblut , E , Perl , S , Kass , N , Ayali , G , Hemsani , E and Perk , S . (2004) . Identification of a novel nephropathogenic infectious bronchitis virus in Israel . Avian Diseases , 48 : 635 – 641 .
- Moore , KM , Bennett , JD , Seal , BS and Jackwood , MW . (1998) . Sequence comparison of avian infectious bronchitis virus S1 glycoproteins of the Florida serotype and five variant isolates from Georgia and California . Virus Genes , 17 : 63 – 83 .
- Nix WA Troeber DS Kingham BF Keeler CL Jr Gelb J Jr (2000) Emergence of subtype strains of the Arkansas serotype of infectious bronchitis virus in Delmarva broiler chickens Avian Diseases 44 568 581
- Parsons , D , Ellis , MM , Cavanagh , D and Cook , JKA . (1992) . Characterisation of an infectious bronchitis virus isolated from vaccinated broiler breeder flocks . Veterinary Record , 131 : 408 – 411 .
- Schikora , BM , Shih , LM and Hietala , SK . (2003) . Genetic diversity of avian infectious bronchitis virus California variants isolated between 1988 and 2001 based on the S1 subunit of the spike glycoprotein . Archives of Virology , 148 : 115 – 136 .
- Ziegler AF Ladman BS Dunn PA Schneider A Davison S Miller PG Lu H Weinstock D Salem M Eckroade R Gelb J Jr (2002) Nephropathogenic infectious bronchitis in Pennsylvania chickens 1997–2000 Avian Diseases 46 847 858