Molecular epizootiology of avian infectious bronchitis in Russia.

Molecular characterization of infectious bronchitis viruses (IBVs) isolated between 1998 and 2002 from chickens in Russia was performed. More than 250 field samples were tested by reverse transcriptase-polymerase chain reaction using two sets of primers corresponding to the most conserved 3'-untranslated region and the most variable S1 gene region of the viral genome. Ninety-one IBV isolates were characterized by phylogenetic analysis of the S1 gene hypervariable region comprising 136 to 558 nucleotides. The major group of isolates (38 viruses) showed very close sequence relationship with strains of the Massachusetts genotype circulating in Russia since the early 1970s. The analysed region of the other 22 Russian IBVs was similar (from 89 to 98% identity) to that from the strains of European genotypes including D274 (nine isolates), 793/B (10 isolates), and B1648, 624/I and Italy-02 (one isolate in each group). Two isolates from very distant geographic locations in Russia (Far East and the European part) clustered together with Chinese strains of QXIBV genotype. None of the remaining 27 Russian isolates showed a close sequence relationship with known IBV strains available in sequence databases. The majority of these variant viruses clustered into the six novel Russian genotypes, often correlating with their geographic location. The remaining five of them were placed outside these unique groups, also representing new genotypes. These data for the first time demonstrated the high genetic diversity of IBV isolates circulating in Russia.

Detection and estimation of avian infectious bronchitis virus antigen by a novel indirect liquid-phase blocking enzyme-linked immunosorbent assay using chicken and rabbit affinity purified immunoglobulins.

An indirect liquid-phase blocking (LPB) enzyme-linked immunosorbent assay (ELISA) using chicken and rabbit affinity purified immunoglobulin G (IgG) has been developed to detect and estimate avian infectious bronchitis virus (IBV) antigen concentration directly in infected allantoic fluid. The method is based on the principle of binding of specific IgG to the test IBV antigen and the assay of unbound IgG on an antigen-coated ELISA plate. The immunoglobulins are chicken N-terminal S2 peplomeric protein-specific IgG isolated by immunoaffinity chromatography on synthetic peptide coupled to CNBr-activated Sepharose 4B or rabbit polyclonal IgG purified from the serum using Protein A Sepharose 4B. The assay detected all tested IBV strains and field isolates propagated in chicken embryos. Signal to noise ratios were calculated from LPB ELISA absorbance units and a diagnostic threshold was established from the signal to noise ratio frequency distribution of samples positive or negative for IBV by virus titration or reverse transcription polymerase chain reaction. The relative sensitivity of the test ranged between 10(5) and 10(6) median egg infectious doses (EID(50)) for chicken IgG and between 10(3) and 10(4) EID(50) for rabbit IgG, depending on the test strain. The assay is simple and takes less than 3 h to perform. It does not require expensive reagents and can be readily adapted to monitor the IBV antigen concentration in allantoic fluids during propagation of vaccine strains or in samples of freeze-dried, live-attenuated IBV vaccines.