A study has been made of the use of chicken tracheal organ cultures for the isolation and assay of avian infectious bronchitis (AIB) virus from both naturally and experimentally infected chickens. Six strains of AIB virus were investigated, 3 of which had been isolated from natural outbreaks of disease. Two of the virus isolations from the outbreaks of AIB were made directly into tracheal organ cultures without passage in embryonated eggs. Organ cultures prepared from 20-day-old embryos were used since they were found to be somewhat more sensitive in virus assay than those derived from chickens of up to 31 days of age. Ciliostasis, which was used as the marker of infectivity, was complete by 3 days after inoculation with each strain of virus examined. Virus could be isolated from both respiratory and non-respiratory tissue in tracheal organ cultures and these cultures were found to be at least as sensitive as 9-day-old embryonated eggs in detecting AIB virus either in pathological material or in serial dilutions. When virus was assayed in both systems, the titres were very similar. It is considered, therefore, that chicken embryo tracheal organ cultures offer a reliable alternative system to embryonated eggs for studying AIB virus.

SUMMARYAvian infectious bronchitis coronavirus (IBV) inactivated by fl-propiolactone induced partial protection of the trachea in up to 40~ of chickens following one intramuscular inoculation 4 to 6 weeks prior to challenge. Retention of an intact tracheal ciliated epithelium 4 days after challenge was the criterion of protection. There was no correlation between protection and serum titres of virus-neutralizing (VN) and haemagglutination-inhibiting (HI) antibody, which were maximal at about 4 weeks after inoculation. Virus from which the S1 but not the $2 (spike-anchoring) spike glycopolypeptide had been removed by urea did not induce protection or VN or HI antibody. Four intramuscular inoculations of monomeric S1 induced VN and HI antibody in two and four chickens respectively. These results indicate that VN and HI antibodies are induced primarily by S1, that intact spikes are a major requirement for the induction of protective immunity and that this property is probably associated with S1.

The antigenic relationships of 24 strains of avian infectious bronchitis virus (IBV) were investigated by serum neutralisation tests performed in chick embryo tracheal organ cultures. The serum dilution that neutralised 100 median ciliostatic doses (CD50) of virus was estimated from the linear relationship between varying concentrations of each virus strain and the neutralisation titre of homologous antiserum; this dilution defined 1 antibody unit. Antisera diluted to contain 20 antibody units were then tested by neutralisation against 1.5--2.5 log10 CD50 of each strain. Clusters of both strains and antisera in turn were established by methods of numerical taxonomy using as measures of resemblance Euclidean distance and correlation coefficient, and by analysis by principal components. These analyses identified a group of 8 similar strains; neutralisation of the remaining 16 strains was slight. Similar results were obtained by classifying antisera, except that a further group of 3 antisera was demonstrated, each having a neutralising capacity for most strains. Implications for vaccine formulation are discussed.

SUMMARYA method is described of assessing cross-immunity in chickens afforded by strains of avian infectious bronchitis virus (IBV), by observing the presence or absence of ciliary activity in tracheal explants prepared from chickens following challenge.Chickens inoculated with the H120 vaccine strain were challenged intratracheally 3 weeks later with the homologous strain or one of seven heterologous strains of IBV. Vaccinated chickens were protected against challenge by the homologous strain (H120) and four other strains (Massachusetts M-41, Connecticut 46, Holte, Iowa 609), but not by one strain (T). The vaccine strain afforded incomplete protection against one further strain (Iowa 97) and less still against another (Gray). All unvaccinated birds were susceptible to challenge by all strains.The method is recommended for use in cross-immunity studies and for assessing strains for use in IBV vaccines. INTRODUCTIONThe extensive use of avian infectious bronchitis virus (IBV) vaccines has provided a generally effective control of such infections in chickens. Nevertheless there are occasional indications that the immunity of a vaccinated flock has been successfully challenged, either wholly or in part, by extraneous field strains of IBV. There is now considerable evidence from neutralisation tests for the existence of antigenic variation amongst IBV strains (Hofstad, 1958;Berry and Stokes, 1968;Dawson and Gough, 1971;Hopkins, 1974;Cowen and Hitchner, 1975;Johnson and Marquardt, 1975;Darbyshire et al., 1979) but, there are also indications that serological relationships determined in vitro in this way have not always been mirrored by the results of cross-immunity studies in chickens (Winterfield and Hitchner, 1962;Hitchner et al, 1964;Raggi and Lee, 1965;Winterfield and Fadly, 1972).

An indirect enzyme-linked immunosorbent assay (ELISA) was used to detect IgG class-specific antibodies to avian infectious bronchitis virus (IBV) in chickens. The technique detected specific antibody with a high degree of sensitivity and was more sensitive than neutralisation or haemagglutination-inhibition methods of antibody assay. The possible application of the technique for diagnosis and research on IBV is discussed.