Foot-and-Mouth Disease Virus: Selection by

Transcription

1 INFECTION AND IMMUNITY, Jan. 1972, p Copyright 1972 American Society for Microbiology Foot-and-Mouth Disease Virus: Selection by Homogenized Calf Kidney Adsorption and Cell Culture Passage Vol. 5, No. 1 Printed In U.S.A. C. H. CAMPBELL Plum Island Animal Disease Laboratory, Veterinary Sciences Research Division, Agricultural Research Service, U.S. Department of Agriculture, Greenport, New York Received for publication 21 July 1971 Foot-and-mouth disease was passaged 20 times by alternately adsorbing the with homogenized calf kidney and propagating the adsorption-resistant fraction of in cell cultures. Passage of the was accompanied by a marked decrease in pathogenicity for 35-day-old mice and a reduction in its ability to adsorb to homogenized kidney from such mice. Adsorption by homogenized calf kidney and infant mouse kidney also decreased with passage, but pathogenicity for cattle and infant mice remained high. The passaged did not seem to change antigenically. When the 20th-passage was serially passaged 10 times in cell cultures, no reversion in pathogenicity for 35-day-old mice was observed. In previous work in which foot-and-mouth disease (FMDV) was exposed to homogenized mouse or calf kidney for 4 hr at 30 C, approximately 5% of the was not adsorbed (3, 4, 5). This residual was relatively resistant to further adsorption by homogenized kidney. In addition, it was found that by a passage process of alternately adsorbing the with mouse kidney and propagating the residual in cell cultures, was obtained which was relatively nonpathogenic for mice (4). This raised the possibility that the adsorption-passage technique might be used to select for a live vaccine. Therefore, since cattle are the chief recipients of FMDV vaccine in endemic areas, the following work was done to study the effect of adsorption passages on characteristics of FMDV when bovine kidney, rather than mouse kidney, was used during the adsorption phase. MATERIALS AND METHODS Virus. The parent FMDV (O-CANEFA-9, Argentina, 1963) had been passaged four times in primary cultures of bovine kidney cells and stored at -70 C. Cell cultures. Primary cultures of bovine kidney cells were used for production and plaque assay of as previously described (1). Adsorption of. Virus was adsorbed by mixing 0.5-ml portions of diluted [approximately 105 plaquejorming units (PFU) I in Leighton tubes with 0.5-ml amounts of a suspension of homogenized calf or mouse kidney in Hanks' balanced salt solution 65 containing 0.5%' lactalbumin hydrolysate (BSS). Control tubes were prepared with 0.5 ml of and 0.5 ml of BSS (3). The tubes were incubated at 30 C. To determine viral adsorption rates, the contents of two tubes with tissue and two control tubes were harvested at 0.5-hr or 1-hr intervals and assayed by the plaque method for unadsorbed (residual) or that had not been heat-inactivated during incubation. Results were expressed as percentages of the amount of added to each tube. Passage of. FMDV was passaged by two procedures referred to as adsorption passage and control passage (4). Each adsorption passage was started by adsorbing the of the previous passage with homogenized calf kidney for 4 hr at 30 C as described above. The contents of several tubes of and homogenized kidney were then pooled and centrifuged at 900 X g for 10 min. The supernatant fluid containing residual was inoculated in 0.5-ml amounts in bovine kidney cell cultures. After incubation at 37 C for 3 hr to permit adsorption, the cultures were overlaid with 12 ml of BSS and incubated at 37 C for approximately 24 hr, when cytopathic effect was about 95% complete. Culture fluid containing the passaged was then centrifuged at 900 X g for 10 min and stored in 2-ml portions at -70 C. The next passage was started by adsorbing this with homogenized kidney. Control passages were made to test for the selective effects of heat inactivation and passage in cell culture. These passages-were begun by incubating the control of the previous passage in BSS for 4 hr at 30 C as described above. The was then diluted 1:10 to reduce the concentration to approximately that of the residual adsorption-passaged. The diluted control

2 66 CAMPBELL INFECT. IMMUNITY was propagated in cell cultures and stored as described for the adsorption passages. Mice. The passaged es were assayed in Swiss mice of the Rockefeller H strain produced at this laboratory. These mice have been given the designation Piadl: M-1(S)CV (11). Unweaned mice of this strain are highly susceptible to FMDV, but at 2 to 3 weeks of age they begin to develop resistance to the (9). The mice were inoculated intraperitoneally with 0.5 log dilutions of, 10 mice per dilution. Mice that were 14 days old or less were inoculated with 0.03 ml; mice older than 14 days received 0.10 ml. All mice were observed for 10 days after inoculation. Viral end points were calculated by the method of Spearman-Karber (8). Tests for pathogenicity. Passaged es were tested for pathogenicity by simultaneous titrations in mice and cell cultures. Pathogenicity was expressed as the number of plaque-forming units of required to equal one median lethal dose in mice (PFU/LD5o). In addition, pathogenicity for 35-dayold mice was judged by the average time of all deaths which occurred during the titration. Pathogenicity in cattle was tested by inoculating Grade Hereford steers intramuscularly in the left shoulder with 2,000 PFU of in a 5-ml dose. One steer was inoculated with each being tested. The animals were observed daily for 1 week for clinical manifestations of foot-and-mouth disease. Experimental procedures. Twenty adsorption passages and control passages were made. Every 5th passage, the es were assayed in cell cultures, 7-day-old mice, and 35-day-old mice. On the basis of these results, pathogenicities of the adsorption-passaged and control-passaged es were calculated and compared. The es were also tested for their ability to be adsorbed by homogenized calf kidney. Viruses of the 20th passages were tested in greater detail. Adsorption rates were compared with homogenized kidney from 7- and 35-day-old mice as well as homogenized calf kidney; To see if the antigenic characteristics of the es had changed during passage, neutralization tests were performed with a constant amount of antiserum specific for the parent and 0.5 log dilutions of in a plaque assay. Pathogenicity was tested simultaneously in mice 7, 14, 21, 28, and 35 days of age to determine the effect of developing age resistance. Pathogenicity of the two es was also tested in steers. Since there were changes in pathogenicity for mice during the adsorption passages, the 20th-passage es were also serially passaged 10 times in bovine kidney cell cultures to see if reversion would occur when adsorption with homogenized kidney was omitted from the passage procedure. Pathogenicity of the from each of these 10 passages was tested in 35-day-old mice. RESULTS Adsorption. Adsorbability of the adsorptionpassaged by homogenized calf kidney decreased with passage (Table 1). By the 20th passage, the was almost resistant to adsorption by this tissue. However, no appreciable change in TABLE 1. Adsorption of passaged FMDV by homogenzized calf kidney Unadsorbed a (%) Adsorptionpassaged Parent a After exposure to homogenized calf kidney for 1 hr at 30 C. adsorbability of the control was observed. Tests of the 20th-passage es demonstrated further that adsorption-passaged was more resistant than control to adsorption with homogenized kidney from both 7- and 35-day-old mice (Fig. 1). Neutralization. Neutralization indexes obtained with antiserum specific for the parent revealed that both of the 20th-passage es were antigenically the same as or very similar to the parent. The log1o values of the neutralization index were 2.99, 2.81, and 2.82 for the parent, the 20th adsorption passage, and the 20th control passage, respectively. Pathogenicity. During the 20 passages, pathogenicity of the adsorption-passaged for 7- day-old mice did not change appreciably (Table 2). Although about twice as much adsorptionpassaged as control-passaged was required to equal 1 LD50 from the 5th passage on, the PFU/LD50 values for both es were very small. In contrast, pathogenicity of the adsorption-passaged for 35-day-old mice, judged both by PFU/LD50 values and time of death, decreased markedly after the 5th passage. By the 20th adsorption passage, over a million PFU of the were required to produce 1 LD50. However, there was no appreciable change in pathogenicity of the control (Table 3). Tests of the 20th-passage es in mice of various ages demonstrated that pathogenicity of the adsorption-passaged was directly related to the age and, presumably, to the degree of resistance of the mice (Table 4). Both the 20th adsorption passage and the 20th control passage produced typical clinical manifestations of foot-and-mouth disease in steers 5 days after inoculation. The clinical response consisted of high temperatures and vesicular lesions on the lips, tongue, and feet. No difference in time of response was observed. Reversion. Reversion of the 20th adsorption passage did not occur during 10 serial cell

3 VOL. 5, 1972 SELECTION OF FMDV Kidney from 7-day-old mice Kidney from 35-day-old mice SO Adsorption-passage 20 S 0 tadsorption-passa*e 20 o 5-0S 0-1 FIG. 1. Adsorption of 20th-passage es by homogenized kidney from 7- and 35-day-old mice. Infectious in decay controls after 5 hr at 30 C = 50 to 60% ofzero-time value. TABLE 2. Pathogenicity of passaged FMD V in 7-day-old mice PFU/LDbo Adsorptionpassaged (logio) Parent TABLE 3. Pathogenicity of passaged FMD V in 35-day-old mice Adsorption-passaged PFU/LDao Avg day PFU/LDso Avg day (logio) of death (logio) of death Parent Porent xpareet Control passage 20 Control passage 20 O Io IS U -I@ 1A.-. I DISCUSSION The results of these experiments furnish additional information on the variability of FMDV and demonstrate that a variant of the population can be selected by the adsorption passage procedure. Although FMDV type 0 was tested in this study and calf kidney was used for the adsorption phase, the results were quite similar to those of previous work with the adsorption passage procedure using homogenized mouse kidney and FMDV type A (4). In both cases, pathogenicity of the adsorption-passaged decreased for adult mice, but there was little or no change for infant mice. The present results also demon- Adsorption time '111'- - hours culture passages as judged by its pathogenicity for 35-day-old mice (Table 5). The continued to be relatively nonpathogenic (approximately Adsorption time - hours TABLE 4. Pathogenicity of adsorption passage 20 and control passage 20 FMDV in mice of various ages Adsorption passage 20 Control passage 20 Age of mice (days) PFU/LD6o Avg day of PFU/LD5o Avg day (logso) death (logio) of death PFU/LD50), whereas pathogenicity of control remained high at about 102 PFU/LD50.

4 68 CAMPBELL INFECT. IMMUNITY TABLE 5. Virus Effect of serial passage in cell cultures on pathogenicity of adsorptiont passage 20 and control passage 20 FMD V in 35-day-old mice PFU/LD5o (logio) at cell culture passage no Adsorption passage 20 Control passage 20 strated that this decrease in pathogenicity was directly related to the age and, presumably, to the degree of natural resistance of the mice. A number of investigators have reported that avirulent strains of several es were not adsorbed by tissues of animals which were affected by virulent strains (7, 10, 12, 13). Although in the present study the reduction in pathogenicity for older mice might have been due to the reduction in adsorbability of the by tissue from such mice, this possibility does not seem likely. Adsorbability by homogenized calf kidney and infant mouse kidney also decreased, yet pathogenicity of the for steers and infant mice remained at a high level. It seems more likely that the reduced pathogenicity for older mice was related to the heterogeneity of FMDV. Previous results indicated that approximately 1 out of 1,000 infant mouse median lethal doses of was infectious for mother mice (2). Possibly, therefore, a relatively small portion of the parent in the present work was pathogenic for 35-day-old mice, and this portion was readily adsorbed by homogenized calf kidney and thus eliminated by the adsorption passage procedure. The importance of adsorption with homogenized kidney in relation to the changes in pathogenicity and adsorbability during the adsorption passages seems clear. In fact, previous work has shown that passage is not necessary to select FMDV that is slightly less pathogenic for infant mice and less adsorbable by calf kidney (5). Such was obtained simply by adsorbing the parent with homogenized calf kidney. Since changes in adsorbability and pathogenicity did not occur during the control passages, there is no reason to believe that the altered characteristics of the adsorption-passaged were due to selection by cell culture passage or by heat inactivation during the 4-hr adsorption period. The reversion experiment indicated that this population was relatively stable in the absence of adsorption with homogenized kidney during the passages. As previously pointed out (6), residual FMDV can be assayed and propagated in primary cultures of bovine kidney cells even though it is relatively resistant to adsorption by homogenized calf kidney. However, experimental results revealed that residual required much longer to adsorb to cell cultures than the parent. The finding that FMDV obtained by adsorption passages was less pathogenic than the parent raises the question as to whether this procedure could be used for obtaining nonpathogenic for a live vaccine. From this standpoint, the fact that the antigenic characteristics of the 20th-passage es were the same as or at least quite similar to those of the parent was encouraging. Unfortunately, pathogenicity of the 20th adsorption-passaged was not reduced for cattle. Perhaps use of a bovine tissue other than kidney for the adsorption phase of the passages would bring about the desired result. ACKNOWLEDGMENTS I thank G. E. Cottral for inoculating the steers, and Frances Bishop and Donald Card for their excellent technical help. LITERATURE CITED 1. Bachrach, H. L., J. J. Callis, W. R. Hess, and R. E. Patty A plaque assay for foot-and-mouth disease and kinetics of reproduction. Virology 4: Campbell, C. H The susceptibility of mother mice and pregnant mice to the of foot-and-mouth disease. J. Immunol. 84: Campbell, C. H Relationship of donor age to adsorption of foot-and-mouth disease by mouse tissues. Amer. J. Vet. Res. 11: Campbell, C. H Virulence, adsorbability, and antigenicity of foot-and-mouth disease selected by adsorption with homogenized mouse kidney. Arch. Gesamte Virusforsch. 26: Campbell, C. H Pathogenicity in mice of foot-andmouth disease selected by adsorption with calf kidney. Res. Vet. Sci. 11: Campbell, C. H Adsorption characteristics of foot-andmouth disease selected by adsorption with homogenized calf kidney. Arch. Gesamte Virusforsch. 34: Eggers, H. J., and A. B. Sabin Factors detennining pathogenicity of variants of ECHO 9 for newborn mice. J. Exp. Med. 110: Finney, D Statistical method in biological assay, 2nd ed., p Hafner Publishing Co., New York.

5 VOL. 5, 1972 SELECTION OF FMDV Graves, J. H., and G. C. Poppensiek Determination of the optimal age range of mice for use in experimental studies with foot-and-mouth disease. Amer. J. Vet. Res. 21: Holland, J. J Receptor affinities as major determinants of entero tissue tropisms in humans. Virology 15: Institute of Laboratory Animal Resouirces, Committee on Nomenclature A nomenclatural system for outbred animais. Lab. Anim. Care 20: Kunin, C. M Virus-tissue union and the pathogenesis of entero infections. J. Immunol. 88: Sabin, A. B Properties of attenuated polio es and their behavior in human beings, p In 0. V. St. Whitelock (ed.), Cellular biology, nucleic acids and es. New York Academy of Sciences, New York.