In the preceding paper of this series we have described an efficient and versatile apparatus for freeze-drying by vacuum sublimation. This apparatus makes it possible to dehydrate biological materials at low temperatures and, by the use of ionization gauges, to determine the end-point of dehydration of material maintained at temperatures ranging down to --80°C. It is so designed that different samples of the same material can be subjected simultaneously to various experimental conditions. Time, rate, and degree of drying have been shown to have a marked influence on the physical properties of lyophilized biological materials other than viruses by Elser, Thomas, and Steffen (1); Greaves and Adair (2); Flosdorf, Hull, and Mudd (3); Greaves (4) and others. Proom and Hemmons (5) have published working details for the freeze-drying preservation of a collection of more than 1500 strains of bacteria. Various methods of freezing, the degree of drying, and the effect of storage were tested by viability counts. Recently, Hutton, Hilmoe, and Roberts (6) have reported on the effects of these factors on the quantitative survival of Brucella abortus. Similar studies dealing with the survival of viruses have not been reported.The present investigations deal with the quantitative survival of influenz a virus suspensions, after repeated freeze-thaw cycles, freezing at various speeds, storage in the frozen state at various temperatures, and vacuum sublimation at various temperatures following different types of preliminary treatment. Subsequent investigations are planned which will be concerned with the effects of these factors on the survival of complex cells.

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Experiments reported in the preceding paper have shown that murine typhus rickettsiae cause a uniformly fatal rickettsial peritonitis in the dba strain of mice, while in other strains, under identical environmental conditions, the mortality is less than 60 per cent. In the course of this work, it became apparent that relatively slight changes in room temperature had a marked influence on the course of the infection. Data showing this effect will be summarized in this paper. EXPERIMENTALIn all, 105 mice of mixed sexes were injected intraperitoneally with light, heavy, or massive doses of murine typhus rickettsiae, and maintained at different temperatures. Some animals were kept in an artificially cooled room in which the temperature varied between 65 ° and 73°F. The remainder of the animals were kept in a room in which the temperature was dependent on climatic variation. Some experiments were done during relatively cool spring weather, during which the temperature range was between 70 ° and 80°F. Other experiments were done during heat waves, with temperatures in the animal room ranging from 85-98°F.Of the 105 mice included in this analysis, only 44 represent parallel experiments. Twenty-two of these were kept at 65-73°F. and the other 22, injected with identical inocula, (brain tissue from dba mice dead or dying after intraperitoneal inoculation), were kept at 85-98°F. (approximate). The mortality in the cooler room was 100 per cent while only 2 of 22 animals in the warm room died. The other 61 mice represent experiments which were not strictly parallel, since several different batches of inoculum were used.Smears were made from the peritoneal cavity of each mouse dying, and animals in which huge numbers of rickettsiae were found, without evidence of secondary infection, were regarded as having died of typhus. RESULTSThe results are summarized in Table I. With massive or heavy doses, intraperitoneally injected dba mice invariably died when kept at 70--80°F. (approximate). Even with light dosage, death occurred in all dba mice injected intraperitoneally and kept at 65-73°F. In a very warm room (85-98°F. approximate) 9 out of 13 mice survived following heavy dosage and 20 out of 22 following light dosage. A definite lengthening of the incubation period and prolongation of life was noted in animals dying in the warm room.

In a previous paper (1), the recovery of a virus morphologically identical with psittacosis from pigeons on a thiamin-deficient diet was reported. This virus, injected intracranially into mice behaved like typical psittacosis, producing fatal meningoencephalitis with typical elementary bodies. The virus differed from typical psittacosis, however, in that intraperitoneal injection in mice failed to produce apparent illness. Cross immunity tests with typical psittacosis were not carried out. The conclusion was drawn largely on morphological grounds that the virus was closely related to, if not identical with that of psittacosis.Francis and Magill (2), in 1938, described a virus recovered from ferrets inoculated with throat washings from human cases of an influenza-like respiratory infection. It was not clear whether this virus was derived from the inoculated human materialor from the ferrets. In mice, it produced fatal meningitis after intracranial injection, and fatal pneumonitis after intranasal injection. Mter intraperitoneal inoculation, a fatal meningitis was produced in some mice. The hepatic necroses characteristic of psittacosis were not found. They were unable to identify their virus, and gave it a descriptive name--the virus of meningopneumonitis.Eaton, Beck, and Pearson (3) have recently reported the recovery of a virus from four cases of human atypical pneumonia by intranasal injection into mice. This virus, in its properties, was practically identical with the virus of Francis and Magill. It showed immunological relationship but incomplete immunological identity with the latter virus and also with psittacosis. It produced a fatal illness in mice, transmissible by intracranial or intranasal inoculation, but like our pigeon virus, was apparently non-virulent in mice when injected intraperitoneally.The experiments to be reported here were designed to test the hypothesis that these viruses might be related to the virus recovered from pigeons.