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%.

A novel infectious bronchitis variant, designated as IS/885/00, associated with nephritis, was isolated from outbreaks in 23 broiler farms in Israel. The virus was first identified by reverse transcriptase-polymerase chain reaction and showed a distinct restriction fragment length polymorphism pattern from previously described Israeli isolates. Sequence analysis of the S1 gene and the deduced amino acid sequence revealed 97.2% protein similarity to genotype IS/ 720/99 and 71.6% similarity to the vaccine strain H120, the only strain permitted for use in this country. A database search in GenBank revealed a closely related isolate from Egypt, Egypt/Beni-Seug/01, with 96.6% similarity. Other published nephropathogenic infectious bronchitis virus strains/isolates shared less than 77% similarity with IS/885/00. A vaccine protection test in specific-pathogen-free chicks indicated 91% protection to the trachea and only 25% protection to the kidneys in vaccinated birds challenged with IS/885/00.

A sensitive and specific method for the diagnosis of infectious bronchitis virus (IBV) is of great importance. In this study the development of a real-time TaqMan RT-PCR targeting the highly conserved nucleocapsid (N) gene of IBV and including an internal PCR control is described. The assay was specific for IBV and did not detect other avian pathogens, including turkey coronaviruses. A comparative limit of detection was determined for M41, an embryo-adapted strain, and IS/885/00, a poorly embryo-adapted variant. For M41 real-time RT-PCR and virus isolation were one or two times more sensitive than RT-PCR targeting the N or spike glycoprotein (S1) genes, respectively. For IS/885/00, real-time RT-PCR was more sensitive by tenfold than virus isolation and 30- or 40-fold than by N gene or S1 gene RT-PCR, respectively. Real-time RT-PCR and virus isolation were 17-75% more sensitive than RT-PCR targeting the S1 gene for testing tracheal swabs directly from experimentally infected chicks. When tracheal and cloacal swabs from clinical specimens were tested directly, 50% more samples were positive by real-time RT-PCR than by the S1 gene RT-PCR. Real-time RT-PCR targeting the N gene is more sensitive than common diagnostic assays, allowing rapid and accurate IBV detection directly from clinical specimens, facilitating differential diagnosis.

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