A possible mechanism for selection of virulent avian influenza A viruses in 14-day-old embryonated eggs

The emergence of virulent avian influenza viruses in poultry is unpredictable. To gain insight into the mechanism of this event, we previously examined the possible role of older (14-day-old) embryonated eggs, in which virulent mutants were preferably selected (Horimoto and Kawaoka, Virology 206: 755-759, 1995). However, it is unknown why virulent mutants replicate predominantly in older eggs. In the present study, we compared protease activities responsible for cleavage activation of the hemagglutinin (HA) in allantoic fluids in 10-day and 14-day-old eggs. In vitro assays showed that the protease activities were stronger in the 14-day-old than 10-day-old eggs. The allantoic fluids with strong protease activity degraded HA. These results indicate that replication of avirulent viruses is hampered in older eggs, while that of virulent viruses whose HAs are activated by other intracellular proteases was not, possibly leading to a replicative advantage for virulent mutants in the older eggs.