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Three Beaudette strains of infectious bronchitis virus (IBV) [two chicken-embryo trachea-culture-adapted (E71CET10 and E71CEK10CET30) and one embryo-adapted (E72)] were propagated in 10-day-old embryonated chicken eggs (CE) to investigate growth and lethality patterns. The E72 and E71CEK10CET30 viruses were similar in growth pattern as to logarithmic phase and maximum virus production, but 95% of the CE mortality with E72 virus occurred between 20 and 32 hours whereas with E71CEK10CET30 60% mortality occurred between 36 and 84 hours. The yield of the E71CET10 virus was minimum, with little lethality.

Five trials were conducted to compare four in vitro methods of isolating avian infectious bronchitis virus (IBV)-direct organ culture of infected tracheal rings (DOC), inoculation of tracheal organ culture (OC), inoculation of chicken embryo, and inoculation of cultured cells. DOC was prepared from tracheas of chickens experimentally inoculated with field samples. In the other methods, pooled tracheal and kidney suspensions were used to inoculate OC, chicken embryos, and cultured cells. IBV was consistently isolated at the initial passage by the DOC and OC inoculation systems, but it was not always isolated by embryo inoculation and never isolated by cultured-cell inoculation. When combined with immunofluorescent staining, DOC was much more efficient than the OC inoculation system for isolation and identification of the five strains of IBV tested because of its simplicity and speed.

Simple assay systems for infectivity titrations of avian infectious bronchitis virus (IBV) in chicken embryo trachea organ cultures (OC) were developed using plastic multiplate wells with one tracheal ring per well; these assays appeared to be much more satisfactory than the conventional rolled-tube method. The medium, 0.05 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-buffered Eagle minimal essential medium was not changed during observation. A medium containing 0.4% bovine serum albumin did not influence the virus yield, but did stabilize virus viability during storage. Reproducibility of results obtained in the OC system was confirmed by performing replicate titrations of the Beaudette strain with three different passage histories. The mean virus titers in the OC were lower than those in chicken embryos, depending on the IBV passage histories. The time required for ciliostasis was related not only to the concentration of virus, but also to the IBV passage history. Application of OC techniques for the constant serum-variable virus neutralization test gave low neutralization indexes with excellent reproducibility as compared with those obtained in the chicken embryo assay system. Also, the slopes of neutralization curves obtained by assays in OC were less steep than those seen in the chicken embryo system.

Sensitivity of the Beaudette strain of infectious bronchitis virus (IBV) to non-antibody inhibitors in neutralization tests depended on the passage history of the virus. The chick embryo kidney cell-adapted (E71 CEK11) virus was the most sensitive, but after one chick embryo (CE) passage (E71 CEK11 E1), this virus showed reversion to the sensitivity of the parent virus (E71). IBV inhibitors in chicken serum could be removed by treatment with trypsin but not with phospholipase C.

The ciliostatic and embryo lethal effect of four strains of avian infectious bronchitis virus (IBV) were observed at two different incubation temperatures. In organ cultures, nephrosis-producing IBV strains (GN-2 and M-41) were slightly more ciliostatic and respiratory strains (Beaudette and Kita-1/Tokushima) less ciliostatic at 40.5 degrees C than at 37 degrees C. Mortality of chicken embryos infected with the GN-2, M-41 and Kita-1/Tokushima strains was greater at 40.5 degrees C than at 37 degrees C, while the reverse was true of the Beaudette strain which caused no embryo mortality at 40.5 degrees C. The Beaudette strain of IBV was the only one which could not be recovered from the kidneys of young chickens four days after infection. These results indicate that the different IBV strains may show variation in virulence at different temperatures in ovo and in organ cultures and this may be a reflection of the virulence and tropism for chickens.