Influenza Virus in Allantoic Sac Tissue Culture

Abstract

Summary The use of allantoic sacs in suspended cell tissue cultures for the growth of influenza viruses has been investigated. Influenza A and B viruses grow readily in such cultures, with either Hanks-Simms' solution or Tyrode's solution as diluents. In the presence of the former solution, viral titers (influenza A) in tissue cultures reach a maximum slightly more slowly and decline more slowly than in Tyrode's solution. Step-wise curves of multiplication of virus are readily demonstrable without recourse to dilution or interference techniques. A complete cycle of multiplication of influenza A virus in this system requires approximately 18 hours, and increases the titer about 100-fold. Frequent changes of the nutrient fluid and the addition of embryo extracts do not increase the capacity of the tissue to support viral growth. The titer of hemagglutinin in such cultures is affected by the amount of tissue in the cultures and by the size of the inoculum. Quantitative study of the pentose and desozy-pentose nucleic acids of the cultures showed a progressive decrease in the former during the incubation of a tissue culture and insignificant alterations in the latter. Viral growth in such cultures may have increased the rate of decline of PNA more rapidly than in uninfected cultures, but had no consistent effect on DNA. Tyrode's and Hanks-Simms' solutions exerted similar effects on nucleic acid concentrations. The incorporation of radioactive phosphorus into nucleic acids of both types by the tissue cultures was rapid. There were no consistent effects on the pattern of uptake of phosphate by the tissue cultures as a consequence of viral multiplication in the cultures. The data indicate that multiplication of tissue cells was not prominent in these cultures, and that a net degradative effect predominated. Uptake of radioactive phosphorus offers a more reliable indication of sufficient viability of tissue cells to support viral growth in suspended cell cultures than do increases in nucleic acid concentration. In the presence of cortisone, both infected and uninfected cultures were characterized by the appearance of numerous cystic blebs of tissue, arising from the culture fragments of allantoic sac. Such outpouchings were less common in control cultures. No consistent effect of cortisone on the concentrations of pentose nucleic acid (PNA) or of desoxypentose nucleic acid (DNA) in the cultured tissues was demonstrable. The rate of incorporation of radioactive phosphate into PNA was slightly accelerated both in infected and uninfected cultures, but incorporation of phosphate into DNA was not significantly altered by the hormone. Cortisone did not affect viral multiplication significantly.