'13: 333233“? 33 533323 3 “3333‘ £30 3333.333 33 3:3 '33 3333321} "T2443 33 3313333 3 5213 33335 33 “$2333 ‘33? 35223 333933 35 33‘s. :3; ‘1‘“ g 1...; .31". flit: 3 N"";"-“ :45». m5; 3.25.3323; ufli-"E: v.1“ S‘q‘ ha 3 3433;? sue. Emu-"3’ .ar‘ . .n 1 "3'3" masxs THE EFFECT OF SUBLIMATION AND STORAGE ON AN EGG-ADAPTED STRAIN OF INFECTIOUS BRONCHITIS VIRUS BF Sally A. Billings A THESIS Submitted to the School of Science and Arts of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1956 Sally A. Billings Abstract 1 Samples of an egg-adapted strain of infectious bronchitis virus in the form of 2 ml portions of infected allantoic fluid were dehydrated by sublimation for 12, 2h and.h8 hours, respec- tively, sealed‘ig 33332 at a terminal pressure of 100 microns, and stored at h° C, 20° C and 37° C for one, three and six months. The samples were titrated in embryonating chicken eggs to determine the effect of sublimation and/or storage on viral activity. The titer of the virus prior to drying was 10-7°2 infective doses per 0.1 ml. After 6 months storage at h° C samples dehydrated for 12 hours had a titer of 10-6.5. Under the same conditions, the titer of samples dried 2n and h8 -6.6 and 10'6‘8, respectivelyo hours was 10 Virus dried for 12 hours and stored at 20° C for 6 months had a titer of 10'5’5. Virus dried for 2h and hB hours had titers of lO'h‘B and 10'5‘7, respectively. After storage at 37° C for 6 months virus dehydrated for 12 and 2h hours was completely inactivated. Samples dried for h8 hours had a titer of 10'1°2. This study demonstrates that infectious bronchitis virus dried as long as h8 hours maintained a slightly higher level of infectivity during 6 months storage. From.a practical view- point, this advantage is not great enough to warrant the additional time involved if large numbers of samples had to be Sally A. Billings 2 processed in a short period of time. If stored at h° C, infectious bronchitis virus dried for 12 hours could be kept for 6 months without an appreciable loss of viral ac- Richard Eugene Billings ACKNOWLEDGMENTS The author wishes to express her sincere appreciation to Dr. C. H. Cunningham for his con- sideration, encouragement and guidance throughout the study. Technical assistance generously given by Mrs. Martha P. Spring is gratefully acknowledged. TABLE OF C ON TENTS PAGE INTRODUCTION 1 REVIEw OF LITERATURE . . . . . . . . . . . . . . . . 2 MATERIALS AND METHODS . . . . . . . . . . . . . . . . 8 RESULTS 12 .DISCUSSION . . . . . . . . . . . . . . . . . . . . 22 SUMMARY . . . . . . . . . . . . . . . . . . . . 25 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . 26 INTRODUCTION This study was undertaken in an attempt to determine the effect of sublimation and storage on an egg-adapted strain of infectious bronchitis virus. REVIEW OF LITERATURE Schalk and Hawn (l93l) in North Dakota were the first to describe infectious bronchitis in chickens. The most characteristic gross lesions in infected birds were found in the lungs and bronchi. The lungs were described as being in a state of acute congestion accompanied by a sero-mucoid exudate in the bronchi and bronchioles. Frequently, a mucoid exudate could be found in the nasal sinuses. Berkefeld filtrates of exudates and tissue extracts were capable of inciting the disease in baby chicks. Beach and Schalm‘ (1936) showed that the disease could be produced by nasal, tracheal, and bronchial exudates passed through V, N, and W Berkefeld filters. Chickens re- covered from.the disease were resistant to further infection but were not immune to laryngo-tracheitis or infectious coryza. They observed that the virus dried by Swift's method and stored in the refrigerator for 180 days was Still capable of producing the disease. The virus remained infective for 80 days when stored in the refrigerator in 50 percent glycerol. Beaudette and Hudson (1937) were the first to cultivate the virus in embryonating chicken eggs. Dwarfing of the embryo accompanied by enlargement of the liver was observed. No lesions were produced on the chorioallantoic membrane which was thinner than normal and adhered closely to the shell membrane. They found that in early passages the virus was relatively non-lethal to the embryo but acquired lethal- ity with continued passage. Delaplane and Stuart (19ul) confirmed the observations of Beaudette and Hudson on the acquired lethality of the virus for chicken embryos inoculated by the chorioallantoic membrane route . Gross lesions consisted of white foci in the liver and congestion and enlargement of the kidneys. The embryos were dwarfed and occasionally hemorrhagic. The virus survived storage in the fresh frozen state in the freezing compartment of an electric refrigerator for four and one-half months and at room temperature for five to seven days. Cunningham and Stuart (l9h6) reported the effect of cer- tain chemicalagents on an egg-adapted strain of infectious bronchitis virus. One part of the virus suspension was mixed with nine parts of the chemical agent, allowed to react for three minutes at room temperature, and injected into eggs via the allantoic sac route. The criterion for inactivation of the virus was survival of the embryo. Phenol, 3 percent and 1 percent, liquor cresolis saponatus, 3 percent and 1 percent, tincture of metaphen, undiluted and 1 percent, potassium permanganate, l-l,000 and 1-10,000, ethyl alcohol, 95 percent, 70 percent, ho percent and 25 percent, Neoprontosil, 5 percent, and formalin, 1 percent produced complete inactivation of the virus. Boric acid, A percent, and tincture of iodine, 0.01 percent were without effect. Cunningham and Stuart (191;?) studied the pH stability of an egg-adapted strain of infectious bronchitis virus. For the first 60 days of the experiment the virus was more stabile in an acid medium than in an alkaline medium. From.60 to 170 days there was a shift to greater stability in an alkaline medium. Virus in a phosphate buffer at a pH of 7.79 remained active for 170 days. Virus in allantoic fluid at a pH of 7.8 was active for 100 days. Undiluted virus at a pH of 8.20 remained active for lh2 days. The criterion for viral stability was death of the embryos. V Cunningham and Stuart (19h?) observed that egg-adapted infectious bronchitis virus stored at -25° C or -70° C had a higher virus activity than did samples stored at -10° C. Freezing and thawing of infectious bronchitis infected allantoic fluid produced two types of precipitates, one soluble at room.temperature and the other insoluble at room temperature. The insoluble precipitate could be deposited by centrifugation without affecting the potency of the virus in the supernatant fluid. No significant differences were noted when Difco nutrient broth, Difco tryptose phosphate broth, 0.85 percent saline, or M/lO phosphate buffer were used as diluents in comparative ti- trations. An egg-adapted strain of infectious bronchitis virus was dried at 0.15 mm mercury pressure for 8 hours, sealed in 32239 and stored at H° C. After 7 days the virus was recon- stituted to volume with 1 ml of sterile distilled water and subsequent titration demonstrated a hundred-fold decrease in virus activity. Delaplane (19H7) reported that infectious bronchitis virus inoculated into embryonating chicken eggs via the allantoic sac route produced dwarfing of the embryo as early as the first passage. Beaudette, Miller, Bivins, and Hudson (l9h8) dried egg- adapted infectious bronchitis virus from.the frozen state over anhydrous phosphorus pentoxide in a vacuum desiccator. They found that samples stored over anhydrous phosphorus pentoxide in an evacuated desiccator in the refrigerator re- mained active up to 68h days. Reagan, Hauser, Lillie, and Craige (l9h8), by means of electron microscOpy, demonstrated the virus to be round with a mean diameter of 90 mu. Filamentous projections were present on some of the particles. Reagen, Brueckner, and Delaplane (1950), using improved techniques, reported the virus to be round with a mean diameter of 70 mn. Filamentous projections similar to those of New- castle disease virus, were observed. Preservation of Other Viruses Olitsky (1939) reported that lyophilized brain tissue infected with avian encephalomyelitis virus was found active after 68 days. Turner and Fleming (1939) observed that influenza virus, PR8 strain, in a 10 percent mouse lung suspension stored at -78° C for approximately 3 years was fatal“ to mice in essen- tially the same dilution as before freezing. Under the same conditions similar results were obtained upon testing the virus of meningopneumonitis in a 10 percent mouse lung suspension. The virus of lymphogranuloma inguinale in a mouse brain suspension remained pathogenic for mice after storage at -78° C for 10 months. ‘Wooley (1939) reported that the viruses of lymphocytic choriomeningitis and St. Louis encephalitis in brain tissue remained viable in storage 378 and 833 days, respectively, after being frozen, dried.;g vacuo, and stored at 5° C. Bauer and Pickels (19h0) found that yellow fever virus, in a medium rich in protein, desiccated at -18° C. and stored at refrigerator temperature was still virulent after 10 years. Hoffstadt and Tripi (l9h6) observed that the viruses of the Levaditi and Cutter strains of vaccinia, herpes simplex, laryngotracheitis, and Rous sarcoma in human serum survived preservation by lyophilization for approximately 3 years with no alteration of characteristics. The 0A strain of Shope's fibroma under the same condi- tions did not survive preservation and storage for approxi- mately 3 years. After lyophilization and storage for approximately three years a human serum suspension of the virus of infectious myxomatosis of rabbits exhibited inconstant viability. MATERIALS AND METHODS The virus used was an egg-adapted strain, VllhD, of infectious bronchitis virus in the form.of infected allan- toic fluid. Nine-day old embryos were used throughout the study. Inoculation was via the allantoic sac. The eggs were trans- illuminated for selection of an area of the chorioallantoic membrane free from large blood vessels about 3 mm below the air cell. A small hole was drilled through the shell without piercing the shell membrane, by means of a mmall drill at- tached to the chuck of an electric motor. Another hole was drilled through the shell over the top of the air cell. Tincture of metaphen was painted over the holes and allowed to dry. The shell membrane over the top of the air cell was punctured with a sterile teasing needle to allow equalization of pressure within the egg when the inoculum.was injected into the allantoic sac and to prevent leakage of the inoculum from the site of injection. After injecting the inoculum, using a B-D Yale l-cc capacity tuberculin syringe, fitted with a 27 gauge, l/2-inch needle, the holes in the shell were sealed with melted paraffin and the eggs returned to the incubator. All incubation was at 99° F in an electric forced-draft incubator. At the time of collection of the allantoic fluid the shell over the air cell was painted with tincture of meta- phen and allowed to dry. This portion of the shell was cracked and removed with sterile forceps. Without removing the shell membrane, the allantoic fluid was harvested using a 5-cc syringe fitted with a 20 gauge, 1 l/2-inch needle. Sixty-three eggs were inoculated using 0.1 cc of inocu- lum per egg. Within eighteen hours after inoculation the eggs were candled. Any deaths that occurred during this period were considered to be due to non-specific causes. After the initial candling, the eggs were candled every hour for eight hours and all dead embryos were refrigerated at h° C. Embryos still living at the end of this period were also refrigerated at h° C. for as long as 12 hours. The allantoic fluid from both living and dead embryos was collected, pooled, and dispensed in 20 ml portions into sterile 30 ml screw cap vials. The vials of infected allan- toic fluid were stored at -30° C until used. At the time of use 60 ml of the allantoic fluid was thawed quickly at room temperature and centrifuged to sedi- ment the precipitate formed on thawing. Two ml portions were placed into sterile 10 ml pyrex ampoules using a sterile 2 ml volumetric pipette. The ampoules were placed in an ice bath until all the fluid was dispensed to minimize any decrease in activity that might be caused by exposure to room temper- ature. The allantoic fluid was shell frozen at -35° C and 10 attached to an Aminco freeze-dry apparatus!’ Groups of samples were dried for periods of 12, 2h and AB hours, respectively. At the end of the drying period the ampoules were sealed in 13233 using a cross-fire gas-oxygen torch. After removal from the apparatus the ampoules were tested for leaks with a Tesla coil and labeled. Samples from the 12, 2h and H8 hour drying periods were stored at H°, 20° and 37° C for comparative quantitative titrations at one, three, and six months. The samples stored at 20° C were placed in a laboratory bench cabinet where they would not be exposed to sunlight. Skinner and Bradish (1954) observed a decrease in infectivity titer of two to five log units when the viruses of influenza, strain B, Newcastle disease, fowl plague, vaccinia, and vesicular stomatitis were exposed to daylight for four hours. To determine the effect of the drying process on viral activity, the infected allantoic fluid was titrated before sublimation. Immediately following drying, the contents of an ampoule of the dehydrated virus were reconstituted to volume with 2.0 ml of sterile distilled water for titration. In all instances serial ten-fold dilutions, 10-1 through 10-8, of the virus sample were made according to the procedure * American Instrument Company, Silver Springs, Maryland. 11 described by Cunningham (1952). The tubes were in an ice bath through the entire procedure. To each tube was added h.5 ml of sterile nutrient broth. To the first tube 0.5 ml of the sample was added using a 2.0 ml serological pipette which was then discarded.- With another pipette the contents of this tube were aspirated and expelled 20 times to insure adequate mixing of the broth and the virus. This constituted the 10"1 dilution of the virus. The same pipette was used to transfer 0.5 ml of this mixture to the second tube. This procedure was followed until all dilutions had been made using a separate pipette for each dilution. Five eggs were inoculated via the allantoic sac using 0.1 cc of inoculum per egg from each virus dilution. The eggs were placed in the incubator and candled 18 hours later. Mortality occurring within this period was considered to be 7 due to non-specific causes and was not included in the data for calculation of viral activity. After the initial candling, the eggs were candled daily for Six days at which time they were discarded. The tubes containing the broth-virus mixture were incu- bated at 99° F for six days. Lack of growth in the tubes was considered to be evidence of bacterial sterility. 12 RESULTS The results of the titrations of the virus are shown in Tables I, II and III and Figures 1, 2 and 3. The results of viral activity for each dilution are expressed as a fraction in which the denominator indicates the number of eggs inoculated, and the numerator, the emp bryo mortality due to viral activity. The 50 percent mor- tality and point, LDSO’ was calculated according to the method of Reed and Muench (1938). The effect of environmental influences was encountered when the dehydrated samples were reconstituted with sterile distilled water for the titration immediately following sub- limation. While the difficulty is not directly related to the primary objective of the present study, it is given as an experience to be avoided by other investigators. Titra- tion of the above samples showed a level of viral activity ' that was below g priori reasoning and at variance with the 100-fold decrease in activity of the same strain of virus as reported by Cunningham and Stuart (l9h7). The distilled water used for recOnstitution of the de- hydrated virus, and the nutrient broth used as the diluent, had been stored in a refrigerator where two previously opened cans of ether were kept. It was considered that the 13 highly volatile ether had been absorbed by the distilled water and/or the nutrient broth and had reduced the viral activity. The results of the titration with ether-contaminated distilled water and nutrient broth are as follows: Titration of Infectious Bronchitis Virus Using Ether-Contaminated.Diluents 10"3 10"“ 10'5 10"6 10-7 12 hours 0/5 0/5 0/5 0/5 0/5 2'4 houra 0/5 0/5 0/5 0/5 0/5 RB hours h/S 5/5 3/5 1/5 l/S Using freshly prepared distilled water and nutrient broth that had not been in contact with ether, the virus was within the expected infectivity range. The results are shown in Tables I, II and III and Figures 1, 2 and 3. These results show conclusively the virucidal properties of ether. Due to the difficulties encountered with the ether con- tamination and the insufficient supply of nine-day old embryos, the titer of the virus samples immediately following sublima- tion could not be determined. Consequently, the titer of the frozen allantoic fluid was used as the basis to which the activity of the virus samples at the storage intervals could be compare d. TABLE I EFFECT OF SUBLIMATION FOR 12 HOURS AND SUBSEQUENT STORAGE FOR 1, 3 AND 6 MONTHS AT h° C, 20° C AND 37° C ON AN EGG-ADAPTED STRAIN OF INFECTIOUS BRONCHITIS VIRUS Virus Dilution LD 10° 10"1 10'”2 10'3 104* 10"5 10"5 10'7 10"8 5° Infectious Bronchitis Virus Prior to Sublimation - 5/5 5/5 5/5 5/5 5/5 5/5 3/5 o/5 10'7“? Time H° C in Months 1 5/5 5/5 5/5 5/5 5/5 3/5 3/5 0/5 - 10-6.2 3 5/5 5/5 5/5 5/5 5/5 5/5 3/3 3/5 - 10-7 4+ 6 5/5 5/5 5/5 5/5 5/5 5/5 3/5 2/5 - 10-6-5 20° C 1 5/5 5/5 5/5 5/5 5/5 3/5 o/5 o/5 - 10 5/5 5/5 5/5 h/h 5/5 3/1: 0/5 0/5 - 10 5/5 5/5 5/5 5/5 5/5 3/5 2/5 1/5 - 10-5-5 1 5/5 5/5 5/5 u/5 U5 1/5 o/5 o/s - 10-3-5 3 5/5 1/5 0/5 0/5 0/5 0/5 0/5 0/5 - 10 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 - . . ,. . . _ . . _ ~ n . _- 4'5: r are s:n_: n. Tut n... a 1- u .. IOeI.J .I .211 -. v -.. ’ .. ’ “.3 ..~~ 3- ‘ In J 3: a 2‘. J in. ‘ .1 I. a _ .ols J'fi:f::4uU8 '5 a .: .. . A, . . .. .- I G ‘ r V, L. . .LJ ' 1.1- .3.1 L) ‘I-L‘ -‘ ‘J .13 if . ) \E a) .15 D C': .'-. 4 a” . , .. . ._~Iav diuldsfluij 4LuiJD“.u TABLE II 16 EFFECT OF SUBLIMATION FOR 2’4. HOURS AND SUBSEQUENT STORAGE FOR 1, 3 AND 6 MONTHS AT h° c, 20° c AND 37° 0. ON AN EGG-ADAPTED STRAIN OF INFECTIOUS BRONCHITIS VIRUS Virus Dilution o -1 -2 -3 41 -5 -6 -7 -8 L950 '10 10 10 10 10 10 10 10 10 ‘ Infectious Bronchitis Virus Prior to Sublimation _7.2 - 5/5 5/5 5/5 5/5 5/5 5/5 3/5 o/5 10 T1? .119. Months 1 5/5 5/5 5/5 5/5 5/5 1/5 o/5 o/5 - 10"” 3 5/5 5/5 5/5 5/5 5/5 5/5 1/5 3/5 - 10-7-2 6 5/5 5/5 5/5 5/5 5/5 5/5 14/11 1/5 - 10""6 20° c 1 5/5 5/5 11/11 5/5 5/5 5/5 1/5 o/5 - 10"6 '4 5/5 5/5 14/5 5/5 1/5 1/5 1/5 o/5 - 10"3’6 5/5 5/5 1/1 5/5 5/5 2/5 o/5 o/5 - 10"“ 8 31° C 1 5/5 5/5 5/5 5/5 2/5 o/5 o/5 o/5 - 10"” 5/5 1/5 1/5 o/5 o/5 o/5 o/5 o/5 - 104°5 0/5 0/5 0/5 0/5 0/5 o/s 0/5 0/5 - fl .1) A”! r' ( Log base 10‘ {n4 0‘1. CD 17 Fig. 2. Effect of sublimation for 2h hours and subsequent storage for 1, 3 and 6 months at h° C, 20° C and 37° C on an egg—adapted strain of infectious bronchitis virus. LL°C 20° 37° c TABLE III 18 EFFECT OF SUBLIMATION FOR 18 HOURS AND SUBSEQUENT STORAGE FOR 1, 3 AND 6 MONTHS AT 1° c, 20° c AND 37° 0 ON AN EGG-ADAPTED STRAIN OF INFECTIOUS BRONCHITIS VIRUS ‘——_____‘—_ _—: —:- Virus Dilution -: T 1"’50 10° 10"1 10‘2 10-3 lo-h 10-5 10-6 10.7 10-8 Infectious Bronchitis Virus Prior to Sublimation - 5/5 5/5 5/5 5/5 5/5 5/5 3/5 o/s 16-7-2 Ti? 1119. Months 1 5/5 1/1 5/5 5/5 5/5 1/5 1/5 0/5 10-6.1 3 5/5 5/5 5/5 5/5 5/5 5/5 2/5 0/5 10-5«8 6 5/5 5/5 5/5 5/5 5/5 5/5 1/5 2/5 10-6-8 20° C 1 5/5 1/1 5/5 5/5 5/5 5/5 3/5 o/5 10-6°2 3 5/5 5/5 5/5 5/5 1/5 1/5 0/5 0/5 1051 6 5/5 5/5 5/5 5/5 5/5 5/5 1/1 0/5 10-5.7 - 413.2. 1 5/5 5/5 5/5 5/5 1/5 1/5 0/5 0/5 10-3.6 5/5 5/5 5/5 0/5 0/5 0/5 0/5 0/5 10-2.5 3/5 3/5 0/5 0/5 0/5 0/5 0/5 0/5 10-1.2 ‘Log base 10 7-0 O\ I P \R <3 .0 19 Fig. 3. Effect of sublimation for AS hours and subsequent storage for l, 3 and 6 months at 4° C 20° C and 37° C on an egg-adapted strain of infectious bronchitis virus. 11°C 20° C 37° C 1...! LA.) .o— .fd) 1.. ti... 1:13 ,a' I) I‘. .J 20 The titer of the allantoic fluid prior to dehydration was 10'7'2. A terminal pressure of 100 microns of mercury was re- corded in all instances. Sublimation for lg:§ours Infectious bronchitis virus dried for 12 hours and stored at h° C for one month showed a titer of 10-6.2. At the third month the titer was lO'Y'h. At the sixth month, or terminal period, the titer was 10-6'5. When stored at 20° C for one month the titer of the virus was 10-S°2. At the third and sixth months the titer was 10'5'5, respectively. A progressive decrease in viral activity was observed when the virus was stored at 37° C. After one month the titer was 10'3'S and after three months it was 10-1.6. The virus was without activity at the sixth month. Sublimation for 2h Hours Infectious bronchitis virus which had been dried for 2h hours and stored at h° C had a titer of 10-5-h at the end of the first month. At the third month the titer was 10"7’2 and at the sixth.month it was 10-6'6. 21 After storage at 20° C for one, three and six.months, -6. - u . the titer of the virus was 10 h, 10 3‘6 and 10 h 8, re- spectively. Storage at 37° C resulted in a decrease in infectivity -7°2 to 10"3'8 at the end of the first month. The third month the titer was lO-1°5. The virus was completely from.lO inactivated by the end of the sixth.month. Sublimation for 48 Hours Virus which had been dried for AS hours and stored at 6.h 1° 0 had a titer of 10" at the first sampling period. By the third month the titer had dropped to 10"5'8 and was 10"6°8 at the terminal period. Virus samples stored at 20° C had titers of 10'6“2 at the first month, and 10'5‘h at the third month. After six months the titer was 10-5.7. After storage at 37° C for one month the titer was 10-3.6. At the third and sixth.months the titer was 10"2'5 and -1.2 10 , respectively. 22 DISCUSSION Three factors must be considered in evaluating the results of this study: the sublumation period, the storage period, and the storage temperature. The storage temperature must, of necessity, be considered in relation to the length of the sublimation period. The amount of heat transferred to the virus particles is related to the moisture present in the dehydrated sample. The more moisture present the greater is the amount of heat transfer and, hence, the more rapid the rate of inactivation of the virus. At all temperatures the samples dried for MB hours maintained a higher level of viral activity than did samples dried for either 12 or 2h hours. The difference is most striking in those samples stored at 37° C. due to the acceler- ation of the reaction between the virus and its environment. It was noted that in samples dried for 12 or 2h hours, a complete loss of infectivity occurred prior to, or during the sixth month of storage. Virus dried for AS hours exhibited a marked decrease in activity but was not completely inacti- vated. The duration of the storage periods played a role in retention of viral infectivity by determining the length of time the virus was exposed to environmental temperatures. 23 This would have become more obvious had the study been ex- tended over a longer period of time. However, using the accelerated reaction at 37° C as an example, the loss of viral activity was related to the duration of the eXposure to the environmental temperature. Similarities were observed between all virus samples titrated after one month of storage at the various tempera- tures. There was a sharp initial decrease in viral infec- tivity presumably due to the effect of the dehydration pro- cess and the temperatures to which the samples were exposed. In samples stored at 20° C and 11° C this initial decrease was followed, in most cases, by a leveling Off of viral activity. As has been stated before, storage at 37° C re- sulted in a progressive decrease in infectivity in all samples. Technical difficulties, such as an error in measurement of the distilled water used for reconstitution of the virus, or improper mixing of the pooled allantoic fluid, may have contributed to the results Obtained in the titrations of samples dried for 12, 2n and AB hours and stored for three months at h° C. No explanation can be given for the inability of the virus dried for 2h hours and stored at 20° C to main- tain a level of activity consistent with that of the other virus samples subjected to the same conditions. From a practical viewpoint, this study serves to point out that although the virus dried for AS hours maintained a alightly higher viral activity than did samples dehydrated for 12 and 2h hours, the difference was not sufficient to warrant the time involved if large volumes of virus were to be dried. Virus dried for 12 hours and stored at h° C re- mained at a high level of infectivity for as long as six months. SUMMARY Infectious bronchitis virus in 2 ml portions was de- hydrated for 12, 2h and RB hours, sealed.in.zggug at a ter- minal pressure of 100 microns, and stored at 4° C, 20° C, and 37° C. One, three and six months after sublimation the virus was titrated in embryonating chicken eggs. The results were expressed as the LDSO‘ After six months storage at u° C, virus samples dehy- drated for 12, 2h and AB hours showed titers of 10'6’5, 10'6‘6 and 10-6'8, respectively. After storage at 20° C for six months, virus samples dried for 12, 2h and NB hours had titers of 10'5°5, low"8 -5.7 and 10 , respectively. Storage at 37° C for six months resulted in a complete loss of viral activity in virus samples dehydrated for 12 and 2h hours. Virus dried for MB hours had a titer of 10-1.2 26 BIBLIOGRAPHY Bauer, J. H., and E. G. Pickels. Apparatus for freezing and drying virus in large quantities under uniform conditions. J. Exp. Med., 71:83-88, l9h0. Beach, J. H., and O. W. Hawn. A filterable virus, distinct from that of laryngotracheitis, the cause of a respira- tory disease of chicks. Poult. Sci., 15:199-206, 1936. Beaudette, F. H., and C. B. Hudson. Cultivation of the virus of infectious bronchitis. J.A.V.M.A., 90:51-60, 1937. Beaudette, F. H., C. B. Hudson, J. A. Bivins, and B. R. Miller. The viability of dried viruses of avian origin. Am. J. Vet. Res., 9:190-l9h, 19u8. Cunningham, C. H., and H. 0. Stuart. The effect of certain chemical agents on the virus of infectious bronchitis of chickens. Am. J. Vet. Res., 7:h66-h69, l9h6. Cunningham, C. H., and H. 0. Stuart. The pH stability of the virus of infectious bronchitis in chickens. Cornell Vet., 37:994103, 1917. Cunningham, C. H., and H. 0. Stuart. Cultivation of the virus of infectious bronchitis in embryonating chicken eggs. M. J. Vet. Res., 8:209‘212’ 1914.7. Cunningham, C. H. ‘A Laboratory Guide for Virology. Burgess Publishing 00., Minneapolis, Minn., 1932. Delaplane, J. P., and H. 0. Stuart. The modification of infectious bronchitis virus Of chickens as the result of propagation in embryonated chicken eggs. Rhode Island Agr. Exp. Sta. Bull., 28h, l9h1. Delaplane, J. P. Technique for the isolation of infectious bronchitis or Newcastle virus including observations on the use of Streptomycin in overcoming bacterial contami- nants. Mimeo. Report, Nineteenth Ann. Pull. Conf., Raleigh, N. 0., June, 19h7. 27 Hoffstadt, R. E., and H. B. Tripi. A study of the survival of certain strains of viruses after lyophilization and prolonged storage. J. Infect. Dis., 78:183- 189, l9h6. Olitsky, P. J. EXperimental studies on the virus of infec- tious avian encephalomyelitis. J. Exp. Med., 70:565- 582, 1939. Reagen, R. L., J. E. Hauser, M. G. Lillie, and A. H. Craige. Jr. Electron micrograph of the virus of infectious bronchitis of chickens. Cornell Vet., 38:190-191, 19h8. Reagen, R. L., A. L. Brueckner and J. Delaplane. Morphologi- cal observations by electron microscopy of the viruses of infectious bronchitis and the chronic res iratory disease of turkeys. Cornell Vet., h0:38h-38£, 1950. Reed, L. J., and H. Muench. A simple method of estimating fifty percent endpoints. Am. J. Hyg., 27 3&93-h97, 1938. Schalk, A. P., and M. C. Hawn. An apparently new respiratory disease of baby chicks. J.A.V.M.A., 31:hl3-h22, l9h1. Skinner, H. H., and C. J. Brandish, Emposure to light as a source of error in the estimation of the infectivity of virus suspensions. J. Gen. Microbiol., 10:377-397, 1951. Turner, T. E., and W. L. Fleming. Prolonged maintenance of spirochetes and filterable viruses in the frozen state. J. Exp. Med., 703629-6379 19.390 Wooley, J. G. The preservation of lymphocytic choriomeningi- tis and St. Louis encephalitis viruses by freezing and drying in vacuo. Publ. Health Repts.. 5h<2h):1077- 1079, 1939. Date Due Demco-293 Thesis Billings, Sally A. The effect of sublima- tion and storage on an egg- adapted strain of infection ‘ ”*— ‘hm-I— g E 131111115. Sally A. ‘ The offset of subli- A nation and storage on an ogg-adapted strain of i infectious bronchitis virus _ _-—~__-, if -1 5": '7 ”$5132 4 .57 N“ H ”I