This paper is a report on the growth and behavior in the embryonated hen egg of certain viruses isolated in the course of yellow fever research in the tropics of Africa and South America. The origin and manner of isolation of the viruses have been described in the first paper of the present series. For purposes of this discussion, the important feature to be borne in mind is that all the viruses were isolated by direct intracerebral inoculation or subinoculation of Swiss mice and had been carried through varying numbers of brain-to-brain passages in these animals.
The objective of this study were: (a) to propagate the viruses in embryonated eggs; (b) to determine the susceptibility of the embryos to infection and the extent and rapidity of the virus increment following inoculation by different routes ; (c) to study tissue affinities of the viruses as reveaIed by titrations and histopathological reactions (the latter to be reported in detail separately ; and (d) to investigate the relative infectiousless of each virus for embryonated eggs and for mice.
With one exception, no great difficulty were encountered in adapting the virus to growth and serial passage in embryonated eggs. The one exception was the Wyeomyia virus, which has thus far resisted cultivation except when inoculated intracerebrally. Five of the viruses, namely Semliki, Mengo, West Nile, Anopheles A and Ilheus, had previously been cultivated in chick embryos by other investigators.
SUMMARY AND CONCLUSIONS
1. Propagation in embryonated hen egg of Ntaya, Bunyamwera, Bwamba, Uganda S, Zika, Anopheles B, Haemagogus A and B, Leucocelaenus, Sabethes an Wyeomyia viruses is reported, and the propagation in the same medium of Semliki, Mengo, West Nile, Anopheles A and Ilheus viruses by other investigators is confirmed.
2. With the exception of Wyeomyia, all of the viruses grew readily and could be maintained in serial passage by yolk sac inoculation using a suspension of the brain or body of the embryo for passage material. However, it was necessary to initiate cultivation of Anopheles B virus by intracerebral inoculation. The Wyeomyia virus was carried through 10 brain-to-brain passages in the embryo and was then lost. It was not infectious to embryos when inoculated by other routes.
3. The multiplication of the viruses, their fatality to embryos and the gross lesions they produce when introduced into the yolk, allantoic and amniotic sacs and when dropped upon the chorioallantoic membrane are described. With the exception of Wyeomyia virus and initiation of the cultivation of Anopheles B virus, inoculations into the yolk and amniotic sacs proved to be the most favorable routes for consistently infecting the embryo.
4. After 3 to 10 passages in the embryo, the majority of the viruses were found to be capable of infecting embryos when introduced into the yolk sac in dilutions as high or higher than those required to produce a fatal infection in mice.
5. Only 4 of the viruses (Semliki, Mengo, West Nile and Ntaya) were consistently fatal to the embryos in high dilutions.
6. Ntaya, Bunyamwera, Bwamba, Uganda S, Anopheles A and B and Ilheus viruses exhibited neurotropism when inoculated into the yolk sac, in that greater concentratione of the infecting agent were observed in the braim than in the body of the embryo.
Semliki, Mengo, West Nile and Zika virus may be regarded as pantropic, as the virus concentrations in the body were equal to if not greater than those in the brain of infected embryos.
7. Haemagogus A and B, Mucocelaenus and Sabethes viruses were consistently found in greater concentrations in the body of the embryo than in the brain. They also exhibited other characteristics in common with each other and with FA mouse encephalitis virus.
8. The implications of these observatiom upon the use of the embryonated egg for the primary isolation of viruses of this category have been briefly discussed.