An experimental analysis is here presented of the conditions that lead to the appearance of non-infectious hemagglutinins (NIHA) in the allantoic fluid of chick embryos injected with standard influenza virus (PR8 strain) which had been exposed to 37°C. in vitro for various periods of time.

On progressive reduction of the infectivity of the undiluted inocula from about 109 to 103 ID50 (103.2 HA units) the yields of infectious virus in 24 hours decreased in straight correspondence 1 millionfold, but those of hemagglutinins only by a factor of 10. Thus the proportions of NIHA in the yields increased sharply but the total quantity obtained decreased gradually.

The quantities of infectious virus produced per ID50 injected were the same throughout this range; i.e., between 50 and 100 ID50, regardless of increasing proportions of heat-inactivated virus in the seeds. This value agrees with previous estimates of yields under other conditions. Thus, initiation and completion of first cycles by the infectious virus remaining in the inocula were not, or at most, slightly inhibited. The inactivated virus, therefore, failed to establish immediate interference. It was capable, however, of holding the infectious process to one cycle. Upon 10-fold dilution of the seeds essentially similar results were obtained except that a slight loss in interfering activity could now be detected with an increase in exposure to 37°C. With further dilutions little or no interference was noted.

The capacity to yield NIHA decreased slowly during exposure of the seeds to 37°C. over a period of 5 days, thereafter more rapidly. It could not be restored by addition of infectious virus. Furthermore, since NIHA was obtained when the seeds contained as little as 102 or 103 ID50, it is unlikely that it was derived from those cells which had adsorbed both infectious and inactivated seed virus. It is suggestive that multiple adsorption of inactivated virus particles per se will yield NIHA. The available information, as discussed, favors the view that the NIHA does not represent seed virus in some form but is newly produced.

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