Further Studies on the Influence of Steroids on

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1 INFECTION AND IMMUNITY, Aug. 1973, p Cpyright ( 1973 American Sciety fr Micrbilgy Vl. 8, N. 2 Printed in U.S.A. Further Studies n the Influence f Sterids n Viral Infectin in Mice D. J. GIRON, P. T. ALLEN, F. F. PINDAK, AND J. P. SCHMIDT Experimental Methds Branch, Epidemilgy Divisin, U.S. Air Frce Schl f Aerspace Medicine, Brks Air Frce Base, Texas Received fr publicatin 2 April 1973 Certain sterids have been reprted t enhance experimental viral infectins, whereas thers have little r n effect. Interference with the interfern system has been suggested as a pssible mechanism fr the viral infectin-enhancing (VIE) activity f hrmnes. In the present study, sterids (prednislne, prgesterne, teststerne) which had n effect n MM virus infectin demnstrated VIE activity against Mayar virus infectin. It is suggested that the VIE activity f a hrmne may be dependent n the viral agent used fr challenge. Data are als presented which suggest that, in additin t interference with the interfern system, the VIE activity f sterid hrmnes may be the result f at least tw ther actins: (i) alteratin f cell membranes; (ii) stimulatin f initial viral replicatin. It was previusly reprted that the sterid hrmnes estrne, crtisne, and hydrcrtisne markedly increased the susceptibility f mice t lethal infectin by MM virus (a member f the encephalmycarditis grup f viruses), whereas prednislne, prgesterne, and teststerne had n effect. Prgeny virus appeared in the sera and brains f estrnetreated animals 48 h earlier than in untreated cntrls (. In the present study, we investigated the pssibility that the bserved virus infectin-enhancing (VIE) activity may be a reflectin f an alteratin f the permeability f the peritneal membranes, permitting the virus t reach target rgans mre rapidly via the vascular system. In additin, the effect f varius sterids n the lethal infectin f mice by Mayar virus (grup A arbvirus) and a cmparisn f the curse taken by MM and Mayar viruses in estrne-treated and untreated mice were determined. MATERIALS AND METHODS Viruses. Stck suspensins f MM and Mayar viruses were prepared and titrated in L cell mnlayer cultures by the methd previusly described (6). The mean lethal dse (LD5) f each virus preparatin in mice was cmputed by the methd f Reed and Muench (9) by using the intraperitneal rute f infectin. In subsequent experiments, each virus was inculated intraperitneally init mice at the dse indicated, and the number f deaths was recrded daily fr 14 days. 151 Animals. Female Swiss albin mice f the same age, weighing 14 t 18 g, were purchased frm Simnsen Labratries, Gilry, Calif. Mice were arbitrarily selected and hused in grups f 1. Sterids. Estrne, prgesterne, hydrcrtisne, prednislne acetate, and teststerne were purchased frm Upjhn Labratries. All sterids, except prgesterne which was in il, were in aqueus slutins as described previusly (. Each hrmne was injected intraperitneally at the dses indicated. Measurement f virus uptake frm peritneal cavity. Twenty-fur hurs after a dse f 2. mg f estrne each, treated mice and nrmal cntrls received 2.7 x 17 plaque-frming units (PFU) f the test virus in.2 ml f Hanks balanced salt slutin (HBS) by the intraperitneal rute. At intervals, mice were decapitated and their bld was cllected in individual tubes cntaining.1 ml f heparin slutin (1, U.S.P. units/ml, Riker Labratries, Nrthridge, Calif.). Peritneal washings, als frm individual mice, were btained by injecting 2 ml f HBS cntaining 1 mg f bvine serum albumin per ml, massaging the abdmen, and remving the fluid. All samples were immediately frzen and stred at -4 C. Just prir t plaque assay, all samples were expsed t tw cycles f rapid freezing and thawing. Statistical analysis. Significance f the difference was determined by chi-square r by t test as indicated. Differences were cnsidered significant if P<.5. RESULTS Uptake f MM virus frm the muse peritneal cavity after estrne treatment. MM virus (2.7 x 17 PFU per muse) was adminis-

2 152 GIRON ET AL. tered t estrne-treated and nrmal cntrl mice, and the virus uptake int the circulatry system and that remaining in the peritneal cavity was measured. The uptake f the virus int the bld is shwn in Table 1. Althugh the differences were nt statistically significant at the.5 level, the data suggest that there was mre virus present in the bld f estrnetreated mice at 5 and 3 min after inculatin than in the bld f cntrl mice. By 6 min, the virus cncentratin was abut the same fr bth grups f animals. Table 2 shws data n the cncentratin f virus in the peritneal washings fr sme f the same mice frm which bld had been taken. At 5 min, there was significantly less virus in washings frm estrnetreated mice than frm cntrl animals. At 3 min, less virus was als recvered frm the treated mice, but the difference was nt statistically significant. At 6 min, virus recvery was abut the same fr bth grups f mice. These data suggest that estrne treatment resulted in an increased disappearance rate f the virus frm the peritneal cavity during the first 3 min after virus inculatin, and are cnsistent with the ntin that the hrmne may have altered the permeability f the peritneal membrane t the virus. Effect f sterids n Mayar virus infectin. Sterid hrmnes (2 mg per muse) were administered t grups f mice 24 h prir t challenge with an LD,b f Mayar virus. The TABLE 1. Mice Cncentratin f MM virus in the bld f individual estrne-treated mice PFU/1-- 4 ml 5a 3 6a Cntrl Avg Estrne Avg a Minutes after intraperitneal inculatin. t prbability that the cntrl and Test estrne grups f values cllected at each sampling time are different is nt significant (P >.5). results (Table 3) shw that treatment with any f the sterids resulted in increased mrtality. In additin, teststerne, crtisne, and estrne significantly decreased the mean survival time. These results cntrast with thse previusly reprted using MM virus as the infectius agent (. In thse studies, prgesterne, prednislne, and teststerne had n effect n mrtality rate r survival time. Replicatin f Mayar and MM viruses in hrmne-treated and untreated mice. The finding that certain hrmnes (prednislne, prgesterne, teststerne) differed in their effect n lethal infectin by MM and Mayar TABLE 2. Cncentratin f MM virus in peritneal washings frm individual estrne-treated mice Mice PFU/1-6 ml 5a 3a 6a Cntrl Avg Estrne Avg pb <.2 NS NS aminutes after intraperitneal inculatin. bt Test prbability that the tw grups f values cllected at each sampling are different. NS, Nt significant (P >.5). TABLE 3. INFECT. IMMUNITY Effect f sterids n Mayar virus infectin in mice Treatment 24 h Mrtality MSTc prir t virus (deaths! PbPI, challengea ttal) (days) HBS 15/ Prednislne 3/ NS Prgesterne 25/ NS Teststerne 26/ Crtisne 38/ Estrne 38/ a Tw-milligram dses f each sterid were injected intraperitneally int mice. bchi-square prbability that mrtality rate differs frm that f cntrl grup. Mean survival time. dt Test prbability that MST differs frm that -f cntrl grup; NS, nt significant (P >.5).

3 VOL. 8, 1973 INFLUENCE OF STEROIDS ON VIRAL INFECTION viruses suggested that, during the initial stages, infectin by these viruses might invlve different target tissues. The fllwing experiment was designed t investigate this pssibility and t determine the effect f estrne n the replicatin f each virus in the varius tissues. Estrne (1 mg per muse) was administered t ne-half f a large grup f mice. Twenty-fur hurs later all f the animals were challenged with either MM r Mayar viruses (2 LD5O). At varius time intervals, 1 mice frm each grup were sacrificed, and the sera, brains, and selected rgans were cllected and pled. The rgans were hmgenized in HBS and centrifuged t remve particulate material. The sera and clarified extracts were then assayed fr virus cntent. Representative prtins nly f these tissues were examined, but the results are calculated and expressed as virus cntent f the entire rgan. Ttal bld vlume was estimated t be 2 ml. The results are given in Tables 4 and 5. In cntrl animals, MM virus was nt fund in significant amunts in any f the tissues tested until 72 h after virus inculatin (Table. Except fr the brain, it apparently replicated nly in the liver and was transiently fund in the ther rgans and bld. After estrne treatment, the virus was readily islated frm several tissues as early as 4 h after virus injectin. By 24 h, virus was fund in all f the tissues tested and apparently replicated in the intestines, liver, spleen, and brain. With the exceptin f the brain where final virus titers in cntrl and test animals were within.5 lg f each ther, much mre virus was prduced in estrne-treated mice. Table 5 gives the data btained when Mayar virus was the infectius agent. They suggest that the initial replicatin f the virus ccurred in the intestines. By 48 h, the virus was fund in all f the tissues tested and apparently replicated in all f them, althugh the virus fund in the bld culd be reflecting the release f the agent frm ther tissues. Estrne treatment stimulated viral replicatin, resulting in the earlier appearance f the virus at higher titers than thse eventually attained in cntrl animals. These data shw that Mayar virus prduces a mre disseminated infectin than des MM virus. Estrne stimulates the replicatin f bth viruses and appears t make previusly resistant tissues susceptible t MM virus. DISCUSSION Previus studies indicate that certain sterids enhance experimental viral infectins, whereas 153 thers have little r n effect (1, 2, 4, 8, 1, 11). The present study, hwever, has shwn that the VIE activity f a hrmne may be dependent n the viral agent used fr challenge. Thus, prednislne, prgesterne, and teststerne, which were previusly reprted t have n effect n MM virus infectin ( were fund t exhibit VIE activity when Mayar virus was the infectius agent (see Table 3). Interference with the interfern system has been suggested as a pssible mechanism fr the VIE activity f a hrmne (5). Since prgesterne and teststerne were fund t have n effect n interfern synthesis (7), it is clear that this is nt the nly mechanism invlved. Other mechanisms can nw be suggested. The permeability f the peritneal membrane appears t be altered, allwing the virus t enter the vascular system and reach target tissues mre rapidly. It als appears that the membranes f ther cells are altered, making them mre susceptible t virus infectin. This effect was evident when MM virus was the infectius agent (see Table. In additin, viral replicatin was stimulated, resulting in the earlier appearance f high-titered prgeny virus. A hrmne culd induce sme r all f the abve changes. Expressin f the VIE activity wuld then depend n the characteristics f the virus. MM virus, fr instance, is a gd interfern inducer, is very sensitive t the actin f interfern (3), and initially replicates in relatively few tissues. When such a virus is used, nly thse hrmnes which interfere with the interfern system wuld be expected t shw VIE activity. Mayar virus, n the ther hand, is sensitive t the actin f interfern but is a relatively pr interfern inducer (unpublished data). During the curse f nrmal Mayar virus infectin, very little interfern is prduced, and it des nt appear t play a significant rle in cmbating the infectin prcess. In additin, Mayar virus initially infects many tissues (see Table 5), resulting in a relatively large amunt f prgeny virus frm its first replicatin cycle which culd vercme pssible interfern actin. With such a virus, the VIE activity f a hrmne wuld nt be dependent n interference with the interfern system. It appears, then, that the VIE activity f sterid hrmnes may be the result f at least three hrmne actins: (i) interference with the interfern system; (ii) alteratin f cell membranes, leading t an increased rate f virus disseminatin and making cells mre susceptible t infectin; (iii) stimulatin f initial viral replicatin. Fr the estrgen-mm virus system, the events leading t VIE activity are fairly

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5 VOL. 8, 1973 INFLUENCE OF STEROIDS ON VIRAL INFECTION 155 TABLE 5. Mayar virus replicatin in estrne-treated and untreated mice Virus titers (PFU/tissue)a at varius times after virus inculatin Tissue 6 h 24 h 48 h 72 h 96 h tl trn Cntrl Estrne Cntrl Estrne Cntrl Estrne Cntrl Estrne" Brain <3 <3 <3 1.8 x x X x x x X 16 Lung <3 <3 <3 8.6 x x x x x x x 13 Liver <3 <3 <3 1.6 x x x x x x X 14 Intestine <3 <3 5.2 x x X x x x x x 14 Spleen <3 <3 <3 2.5 x x x x x x x 14 Kidney <3 <3 <3 9.4 x x x x x x x 14 Bld <3 <3 3.4 x x x x x x x 14 NDC a See Table 4 fr prcedure. h Seven f 1 mice survived fr 96 h. By 12 h, there were n survivrs in the estrne-treated grup. C ND, Nt dne. clear. After estrgen treatment, the virus is quickly taken up by the vascular system and transprted t tissues which are nw mre susceptible t virus infectin. At this stage, the interfern system is essentially nnfunctinal s the virus escapes this line f defense. Virus replicatin is stimulated, allwing the viral agent t quickly reach the prper threshld levels fr brain infectin. We are currently investigating the mechanism(s) invlved in the stimulatin f virus replicatin by using an in vitr hrmne-cell system. ACKNOWLEDGMENTS We thank J. Cnnell, R. Ibarra, and K. Flexer fr their excellent technical assistance. LITERATURE CITED 1. Aycck, W. L A subclinical endcrinpathy as a factr in autarcelgic susceptibility t plimyelitis. Endcrinlgy 27: Friedman, S. B., L. J. Grta, and L. A. Glasgw Differential susceptibility f male and female mice t encephalmycarditis virus: effects f castratin, adrenalectmy, and administratin f sex hrmnes. Infect. Immunity 5: Girn, D. J Rle f interfern in the prpagatin f MM virus in L cells. Appl. Micrbil. 18: Girn, D. J., and P. T. Allen Effect f estrgen and ther sterids n MM virus infectin in mice. Infect. Immunity 2: Girn, D. J., P. T. Allen, F. F. Pindak, and J. P. Schmidt Inhibitin by estrne f the antiviral prtectin and interfern elicited by interfern inducers in mice. Infect. Immunity 3: Girn, D. J., and F. F. Pindak Prpagatin f MM virus in L cells. Appl. Micrbil. 17: Girn, D. J., J. P. Schmidt, and F. F. Pindak Effect f prgesterne and teststerne n interfern prductin and n the viral infectin-enhancing activity f estrne and hydrcrtisne. Infect. Immunity 4: Kilburne, E. D., and F. L. Hrsfall Lethal infectin with cxsackie virus f adult mice given crtisne. Prc. Sc. Exp. Bil. Med. 77: Reed, L. J., and H. Muench A simple methd f estimating fifty percent endpints. Amer. J. Hyg. 27: Sprunt, D. H., S. McDearman. and J. Raper Studies n the relatinship f the sex hrmne t infectin. J. Exp. Med. 67: Swartzman, G Enhancing effect f crtisne upn plimyelitis infectin in hamsters and mice. Prc. Sc. Exp. Bil. Med. 75: