Direct Isolation of Human Influenza Virus in Tissue Culture Medium and on Egg Membrane

Abstract

Throat washings in Tyrode's solution were obtained on the second day of illness from a patient acutely ill with influenza. The material was centrifugalized at 2500 revolutions per minute for 30 minutes. The supernatant fluid was then filtered through a graded collodion membrane of 500 mμ average pore size. 1 Flasks containing 4.5 cc. of chick embryo Tyrode medium 2 were inoculated with 2.5 cc. amounts of the filtrate (shown by ferret inoculation to contain active virus). Transfers of 0.5 cc. amounts of the culture material to 4.5 cc of fresh medium were made at 48-hour intervals. Mice were inoculated intranasally with culture fluid of the 5th transfer. Mouse passages were made at 4-day intervals. No significant lesions were seen in the lungs of mice of the first 3 passage groups. In mice of the 4th passage, however, sugestive lesions were seen; these were more definite in the 5th passage, and in the 6th passage death of the mice with extensive pulmonary involvement occurred. The virus was identified as human influenza virus by means of neutralization tests with known immune serum.

These results indicate that virus was recovered directly from the throat of a human influenza patient by the introduction of the filtered throat washings of the patient into tissue culture medium. The virus not only survived but probably multiplied, since the final dilution of the original material was approximately 1:300,000 at the time the culture material was first given to mice. The behavior of the virus after its inoculation into mice was very similar to that of virus established directly in mice from human throat washings. 3

Burnet has reported in detail the behavior of human influenza virus introduced after passage through ferrets, onto the chorioallantoic membrane of the developing chick. The direct cultivation of the virus of common cold on the chorio-allantoic membrane has also been repeated. Attempts were therefore made to utilize this procedure in the isolztion of virus directly from the human patient.