Isolation and characterization of avian coronavirus from healthy Eclectus parrots (Eclectus roratus) from Indonesia.

BACKGROUND AND AIM: Avian coronavirus has a wide range of hosts, from chickens and turkeys to wild birds. This virus causes an economically and, possibly, environmentally, important loss in the poultry industry. Therefore, research into the avian coronavirus in various species of birds is required. The Eclectus parrot (Eclectus roratus) is an endemic bird to Indonesia and Northern Australia and often kept as pets. At present, there has been limited information about avian coronavirus infection among birds. This study aimed to determine the presence of and to characterize avian coronavirus isolated from Eclectus parrots in Indonesia. MATERIALS AND METHODS: Cloacal swab samples were taken from 10 healthy Eclectus parrots (E. roratus). Each isolate was propagated into specific pathogen-free embryonated chicken eggs. The presence of avian coronavirus was determined using three sets of primers targeting the 3' untranslated region (3'-UTR) of avian coronavirus (UTR41+/11-), the N gene of the infectious bronchitis virus (IBVN+/-), and the S1 gene of the IBV (XCE2+/XCE2-). The infectious bronchitis vaccine strain H120 was used as a positive control. Resulting positive bands were sequenced for the S1 gene. RESULTS: None of the isolates was positive for the 3'-UTR, four isolates were positive for the N gene of infectious bronchitis, and two isolates were positive for the S1 gene of the IBV. However, only one isolate (parrot/Indonesia/BX9/16) was sequenced for the partial S1 gene with primers XCE2+/XCE2-. The partial nucleotide sequence of this isolate showed 100% homology with the IBV GI-13 lineage, specifically with a field isolate of the 4/91 variant 1 Israel and the 4/91 vaccine on the hypervariable region 3 site of the S1 gene. CONCLUSION: An IB-like avian coronavirus was isolated from healthy Eclectus parrots. Our results indicate that IBV has a wide range of hosts, which prompt the need to understand the interspecies connection of this virus better.

New criteria for immunofluorescence assay for Q fever diagnosis in Japan.

A study was made to evaluate the cutoff value of indirect immunofluorescent-antibody (IFA) test for Q fever diagnosis in Japan. We used 346 sera, including 16 from confirmed Q fever cases, 304 from Japanese pneumonia patients, and 26 from negative cases. Thirteen sera from the confirmed Q fever cases with an immunoglobulin M (IgM) titer of > or =1:128 and/or IgG titer of > or =1:256 by the IFA test were positive by both enzyme-linked immunosorbent assay (ELISA) and Western blotting assay (WBA), whereas 298 sera from pneumonia patients and 26 negative sera with an IgM titer of < or =1:16 and an IgG titer of < or =1:32 by the IFA test were negative by both ELISA and WBA. In the proposed "equivocal area," with an IgM titer of > or =1:32 and < or =1:64 and/or an IgG titer of > or =1:64 and < or =1:128, we found 9 sera, 3 from confirmed Q fever cases and 6 from Japanese pneumonia patients, by the IFA test. Three sera from the confirmed Q fever cases and one of the sera from pneumonia patients were IgM and/or IgG positive by both ELISA and WBA. These results suggest that a single cutoff value for the IFA test may cause false-positive and false-negative results. In conclusion, this study showed that an "equivocal area" should be used for the IFA test rather than a single cutoff value and that sera in the equivocal area should be tested by additional serological assays for confirmation.

Anti-idiotypic antibody specific for an infectious bursal disease virus-neutralizing monoclonal antibody does not interfere with the virus infection.

Infectious bursal disease virus (IBDV) capsid protein, VP2, contains a hypervariable domain that is recognized by virus-neutralizing antibodies. The virus-neutralizing epitope is highly conformation-dependent and the domain is speculated to be involved in the virus-target cell interaction. In this study, a polyclonal anti-idiotypic antibody (anti-id) was generated by the sequential immunization of a rabbit with a virus-neutralizing monoclonal antibody GI-11 which recognizes the VP2 hypervariable domain. Although the anti-id, which mimics the conformational epitope in the VP2 hypervariable domain, was expected to inhibit the virus infection, the anti-id did not interfere with either the virus binding or the infection to the target cell.

Isolation of monoclonal antibodies that inhibit the binding of infectious bursal disease virus to LSCC-BK3 cells.

Three hybridoma cell lines producing monoclonal antibodies (MAbs) against LSCC-BK3 cells which are susceptible to infectious bursal disease virus (IBDV) infection were produced and characterized. The MAbs, designated T7, Q11 and Q13, inhibited the attachment of IBDV to LSCC-BK3 cells. Furthermore, these MAbs bound to LSCC-BK3 but not to nonpermissive cells in flow cytometry. MAb T7 detected a 110-kDa membrane protein of LSCC-BK3 cells, whereas Q11 and Q13 reacted with membrane proteins of molecular weights 58-, 85-, 90- and 110-kDa. These observations imply that the 110-kDa protein recognized by all the MAbs is associated with IBDV binding. The MAbs established in this study are useful for studying the interaction between IBDV and its target cell.

Detection of cell membrane proteins that interact with virulent infectious bursal disease virus.

To detect the molecules that interact with infectious bursal disease virus (IBDV), the chicken B lymphoblastoid cell line, LSCC-BK3, which is permissive for virulent IBDV infection was investigated. The sodium dodecyl sulfate-solubilized plasma membrane fraction from the cells was subjected to a virus overlay protein binding assay. The IBDV specifically bound to proteins in LSCC-BK3 plasma membranes with molecular weights of 70, 82 and 110 kDa. This is the first report to demonstrate cellular molecules that interact with virulent IBDV.

Infectious bursal disease live vaccines: changes in the virus population during serial passage in chickens and chicken embryo fibroblast cells.

Two attenuated infectious bursal disease virus strains used as commercial live vaccine were passaged five successive times in specific-pathogen-free chickens and chicken embryo fibroblast (CEF) cells. Both attenuated strains increased in virulence during the passage in susceptible chickens as evidenced by the decrease in bursa/body weight ratios. A direct nucleotide sequence analysis of the VP2 hypervariable domain amplified by the reverse transcription-polymerase chain reaction revealed that the nucleotide at position 890 (T) in both strains was A after the passage in chicken. In addition, the nucleotide at position 890 (A) was T or C after the subsequent passage in CEF cells. Because of the nucleotide differences, the amino acid residue at position 253 (His) in both vaccines was Gln after the passage in chickens, and the amino acid residue Gln was changed back to His during the subsequent passage in CEF cells. The digestion of the amplified fragment with restriction endonucleases Stul and Ncol, which recognize the sequence difference at position 890, showed that the population of the virus that had amino acid Gln at position 253 was gradually increased during the passage in chickens. Conversely, the population of the virus that had amino acid His at position 253 was gradually increased during the subsequent passage in CEF cells.

Seroprevalence of infectious bursal disease virus in free-living wild birds in Japan.

Serum samples collected from 739 free-living wild birds of 44 species from Gifu, Mie and Hyogo Prefectures in Japan during the period 1989 to 1997 were tested for antibodies to infectious bursal disease virus (IBDV) serotypes 1 and 2 by a virus neutralization test. Serological evidence of infection with serotypes 1 and 2 was found in 15 (2%) of the sera of 6 species and 36 (4.9%) of the sera of 11 species, respectively. Antibodies to IBDV were detected from both sedentary and migratory species. These findings suggest that free-living wild birds have an important role in the natural history of IBDV. These findings raise the possibility that the IBDV prevalent in the breeding grounds of these birds in other countries could be imported by the migratory species. This is the first report of an extensive serological survey of IBDV in wild birds.

Some characteristics of a cellular receptor for virulent infectious bursal disease virus by using flow cytometry.

A flow cytometric virus binding assay that directly visualizes the binding of infectious bursal disease virus (IBDV) to its target cells was established. The chicken B lymphoblastoid cell line, LSCC-BK3, which is permissive for IBDV infection, bound high levels of the virus. Another B lymphoblastoid cell line, LSCC-1104-B1, bound low levels of the virus, although it was nonpermissive. No virus binding was detected in nonpermissive T lymphoblastoid cell lines. In the binding assay to heterogeneous cell populations of chicken lymphocytes, IBDV (a highly virulent OKYM strain) bound to 94% cells in the lymphocytes prepared from the bursa of Fabricius, 37% cells in those prepared from the spleen, 3% cells in those prepared from the thymus, and 21% cells in those prepared from the blood. Most of the cells, which bound the virus, were surface immunoglobulin M (SIgM)-positive, but a small number of them were SIgM-negative. Additionally, the binding of IBDV to the LSCC-BK3 cells was affected by treatment of the cells with proteases and N-glycosylation inhibitors. These findings may indicate that the IBDV host range is mainly controlled by the presence of a virus receptor composed of N-glycosylated protein associated with the subtle differentiation stage of B-lymphocytes represented mostly by SIgM-bearing cells.