Isolation of Bovine Parainfluenza-3 Virus in Chick Embryos.

Myxovirus parainfluenza-3 (SF-4 bovine strain) was first isolated in tissue cultures of embryonic bovine kidney (EBK) cells (Reisinger, Heddleston, and Manthei, J. Am. Vet. Med. Assoc. 135:147, 1959). This agent is antigenically and biologically related to the human HA-1 strain isolated previously by Chanock et al. in monkey kidney cells (Abinanti and Huebner, Virology 8:391, 1959; Abinanti et al., Proc. Soc. Exptl. Biol. Med. 106:466,1961). At present, both viruses are isolated and propagated in tissue cultures, and tissue-culture products are employed for most research purposes. This investigation indicates that bovine parainfluenza-3 virus can be isolated and produced in 14-day-old embryonated hen's eggs, and that the developing chick embryo is highly susceptible to parainfluenza-3 virus infection. Isolation of parainfluenza-3 virus in chick embryos was compared with isolation in tissue cultures. EBK tissue cultures, maintained in Earle's balanced salts (BSS) with 5.0% calf serum and 0.5% lactalbumin hydrolysate (Nutritional Biochemicals Corp., Cleveland, Ohio), were inoculated with 0.2 ml of nasal-swab material (nasal swabs suspended in 4.0 ml of BSS) from 14 calves with signs of shipping fever. After incubation at 36 C for 6 days, fluids from cultures not showing cytopathic effects were subcultured in fresh cells, and the remaining monolayers were examined for noncytopathic virus by the adsorption-hemagglutination (hemadsorption) test of Vogel and Sholokov (Science 126:358, 1957). Infectious fluids from positive cultures were retained for parainfluenza-3 virus identification. Embryonated hen's eggs (14 days old) were inoculated by the amniotic route with 0.1 ml of nasalswab material. After 4 days at 36 C, the amniotic fluids were harvested and tested for virus by inoculation of EBK cells. Parainfluenza-3 virus was identified by serum-neutralization tests in tissue cultures with immune rabbit serum prepared against the original SF-4 bovine virus isolate. Parainfluenza-3 virus was isolated from six nasal-swab suspensions in both EBK cells and 14-day-old chick embryos. Five embryonated eggs and an equal number of tissue cultures were inoculated with each suspension and, in all cases, eggs were infected more frequently than were corresponding tissue cultures inoculated with larger doses of the same material. The isolation of parainfluenza-3 virus from two samples was complicated in tissue cultures by the presence of infectious bovine rhinotracheitis (IBR) virus which also produces cytopathic effects in EBK cells. IBR virus did not interfere with the isolation of parainfluenza-3 virus in embryonated eggs and was not demonstrated in amniotic fluids 4 days after virus inoculation. Noncytopathic virus, demonstrable by hemadsorption, was not isolated. When three nasal-swab samples containing parainfluenza-3 virus were diluted 1:2 and 1:20 in BSS, virus was isolated from the lower concen-