Recovery in tracheal organ cultures of novel viruses from patients with respiratory disease.

Despite recent advances in tissue culture methodology, only 20 to 35 per cent of adults with acute upper respiratory tract illness yield viruses which can be cultivated by the standard virus recovery techniques.3' 4, 12 Recently, an increase in virus isolation from patients with common colds was achieved by Tyrrell and Bynoe through the use of human embryonic tracheal and nasal organ cultures.9' 20 Viruses were detected in harvests from organ cultures either by the induction of hemadsorption or cytopathic effect (CPE) in subinoculated tissue culture or by the production of colds in volunteers. One virus, strain B814, which was recovered by these techniques and produced colds in volunteers, was of particular interest since it was ether-labile and grew only in human ciliated epithelium. Subsequently, Tyrrell found that this virus resembled in morphology the avian infectious bronchitis virus (IBV), a medium-sized virus which appears to be distinct from the myxoviruses.2, 18 Another newly recognized, medium-sized, ether-labile virus, strain 229E, was recovered from students with colds by Hamre and Procknow using standard tissue culture techniques.5 During a survey of acute upper respiratory illness which was carried out in our laboratory, and in which the standard tissue culture techniques were used, we noted a sharp decrease in the rate of virus isolation during the winter of 1965-66, similar to that reported by others.3' 4 12 Stimulated by the findings of Tyrrell and Bynoe, we used the organ culture technique to study specimens, from this and a previous survey,12 which failed to yield agents in standard tissue cultures. From 23 such specimens 8 agents were recovered, including 6 with an unusual-morphology which resembled that of IBV. The recovery and properties of these 6 viruses form the basis of this report. Materials and Methods. -Collection of specimens and virus isolation attempts in tissue culture: Specimens were obtained from employees of the National Institutes of Health with acute upper respiratory illness on or before the fourth day of illness.12 Two-tenths ml of freshly collected nasopharyngeal wash fluid was inoculated into each of two tubes of the following tissue cultures: HEp-2, primary human embryonic kidney, rhesus monkey kidney, and human diploid cell strains (HDCS) WI-26, WI-38, and AT-39.13 The remaining fluid was stored at -60C. Tissue cultures were obtained from commercial sources, maintained as previously described,' and were incubated at 330C on roller drums. The tubes were observed for cytopathic effect twice weekly. Monkey kidney cultures were tested for hemadsorption at 5to 7-day intervals, and a single blind passage of most human embryonic kidney cultures was made at 21 days. Only those nasopharyngeal washings in which virus could not be detected by these methods were examined by organ culture techniques. Acute phase sera were drawn at the time the washings were collected and convalescent phase sera approximately 3 weeks thereafter. Organ cultures: Human embryonic tracheal organ cultures were prepared and maintained by a modification of the method of Hoorn and Tyrrell.6,7 Tracheas were obtained from fetuses spontaneously aborted at 5-9 months' gestational age. The tissue was excised en bloc by sterile technique and immediately stored in cold Hanks' balanced salt solution with 10% fetal calf serum,