J. gen. Virl. (1982), 63, 325-332. Printed in Great Britain 325 Key wrds: herpes simplex virus/interfern inductin/genetics f hst resistance~x-linkage An X-linked Lcus Influences the Amunt f Circulating Interfern Induced in the Muse by Herpes Simplex Virus Type 1 By RAINER ZAWATZKY, I* HOLGER KIRCHNER, 2 JAQUELINE DEMAEYER-GUIGNARD ~ AND EDWARD DEMAEYER 1 11nstitut Curie, Universit~ de Paris-Sud, Orsay, France and 2 Institute f Virlgy, German Cancer Research Center, Heidelberg, Federal Republic f Germany (Accepted 15 July 1982) SUMMARY Prductin f circulating interfern (IFN) was measured in inbred muse strains fllwing intravenus injectin f herpes simplex virus type 1 (HSV-1). IFN titres reached maximal levels 2 t 3 h after injectin f virus and a 10-fld difference was fund between C57BL/6 mice and BALB/c, as high and lw prducers respectively. Mendelian analysis revealed that HSV-induced IFN prductin is gverned by several lci, ne f which is X-linked. The strain distributin pattern btained frm results in recmbinant inbred lines and the results btained in the cngenic B6-C-H-28c-If-11 strain furthermre indicated an absence f clse linkage t If-1. It is cncluded that the levels f HSV-induced early IFN prductin are influenced by several autsmal lci and ne X-linked lcus. INTRODUCTION Several genetic lci in the muse influence the levels f circulating interfern (IFN) prductin in respnse t viruses. The lci cntrlling IFN prductin with ne virus d nt necessarily have an effect n interfern levels induced by anther virus; fr example, bth Newcastle disease virus (NDV) and Sendai virus induce 10-fld higher levels f serum IFN in C57BL/6 than in BALB/c mice but segregatin analysis and the use f recmbinant inbred (RI) muse strains have shwn that different lci are invlved in regulating IFN prductin in respnse t the tw viruses (DeMaeyer & DeMaeyer-Guignard, 1979). Injectin f herpes simplex virus (HSV-1) int mice als results in high levels f circulating IFN, and it culd again be shwn that the amunt f IFN prduced is influenced by the gentype f the hst, in that C57BL/6 mice prduce higher amunts f IFN than mice f ther strains such as DBA/2 r BALB/c (Zawatzky et al., 1981). It was therefre f interest t determine the number f lci invlved in the quantitative difference between high and lw IFN respnders t HSV, and the linkage f these lci t ther If-lci. In this paper, we present evidence that early (between 2 and 3 h) IFN prductin in respnse t HSV is under the quantitative cntrl f at least three lci, ne f which is X-linked. METHODS Virus. Herpes simplex virus type 1, strain WAL (HSV-1 WAL), derived frm a virus stck as previusly described (Kirchner et al., 1978), was plaque-purified and prpagated in RITA cells (a cntinuus line f mnkey kidney rigin screened fr the presence f mycplasma) in Medium 199 (Mrgan et al., 1950), supplemented with 5 % fetal bvine serum (FBS) at a multiplicity f infectin (m..i.) f 0-02. Virus particles were recvered 24 h later frm the superuatant f RITA cells by sedimentatin at 27000 g fr 1 h. Extractin and purificatin f infectius virus DNA and subsequent infectin f BSC-1 ceils (African green mnkey kidney cells) were perfrmed essentially as described by Graham et al. (1973), using the calcium phsphate precipitatin technique. Plaques f HSV-I culd be identified 3 days after infectin f BSC-1 cells with virus DNA. Virus was then prpagated again in RITA cells at lw m..i. (0.02) t btain a new virus stck. Derived frm the latter, the first passage at high m..i. (1-5) was used in all the experiments f this study. The titre f this preparatin was 108 p.f.u./ml. It will be referred t belw as HSV. Interfern inductin. Mice received an intravenus injectin, int the retrrbital sinus, f 0.1 ml HSV 0022-1317/82/0000-5283 $02.00 Dwnladed 1982 SGM frm www.micrbilgyresearch.rg by
326 R. ZAWATZKY AND OTHERS suspensin, crrespnding t 107 p.f.u./animal. Bld was drawn between 2 and 3 h later, frm the same site, and after vernight strage at 4 C, the serum was separated and stred at - 20 C. Befre the interfern titratins, all sera were pre-diluted 1 : 10 in minimal essential medium (MEM) and acidified t ph 2 by the additin f 0.5 M- HCI. Fur h f acid treatment was fund sufficient t inactivate any HSV remaining in the serum. The decisin t measure IFN 2 t 3 h after inculatin f the virus was based n the bservatin, frm preliminary experiments, that ptimal IFN inductin was achieved at that time. Interfern titres reached maximal levels 2 t 3 h fllwing virus inculatin, and n difference in kinetics f IFN prductin between parental strains (male C57BL/6 and BALB/c) culd be bserved. Titres remained at maximal levels fr 6 h after virus infectin and then declined slwly. N IFN prductin was bserved in either C57BL/6 r BALB/c mice fllwing intravenus injectin f supernatants frm uninfected RITA cells. Interfern assay. All IFN titratins were perfrmed using a micrtitre assay in 96-well tissue culture plates (Falcn). Apprpriate twfld dilutins f the acid-treated sera were incubated vernight in the presence f muse L cells. The challenge virus, cnsisting f encephalmycarditis virus (EMCV), was then added and the cell prtectin read 24 h later. Each serum was titrated in duplicate. One unit is defined as the reciprcal f the last serum dilutin cnferring at least 50 ~ prtectin against destructin f the L cells by EMCV, and crrespnds t 3 internatinal reference units, based n cmparable titratins f NIH muse interfern preparatin with reference number G-002-904-51 h All units are expressed as lgl0 values. Mice. The inbred strains used in this study were riginally btained frm D. Bailey at the Jacksn Labratry, Bar Harbr, U.S.A., and have been maintained in Orsay, by brther-sister mating fr several years. All mice were 2 t 3 mnths ld at the time f the experiments. Statisticalanalysis f the data. The lg10 values f the titres were used fr the analysis by Student's t-test (Dixn & Massey, 1969), since it has been shwn previusly that values fr circulating IFN levels in mice have a clse t nrmal distributin when pltted n a lg~0 scale (DeMaeyer & DeMaeyer-Guignard, 1979). This was cnfirmed in the present study, as can be seen in Fig. h In additin, the results were analysed nn-parametrically by the Wilcxn rank sum test (Hllander & Wlfe, 1973). In general, there was a gd crrelatin between the utcmes f the tw tests. RESULTS Interfern titres in parental strains (BALB/c and C57BL/6) The mean titres in BALB/c and C57BL/6 mice differed by a factr f abut 10. When males and females were analysed separately, the average titre f the females, bth fr BALB/c and C57BL/6, was slightly higher than the average titre f the males. Fr bth strains, the difference between males and females was statistically significant (P < 0-005) (Table 1, Fig. 1). F1 generatin When IFN titres f F1 prgeny f reciprcal crsses were analysed, a striking difference between males and females was bserved. Fr bth crsses, BALB/c x C57BL/6, and C57BL/6 x BALB/c, the titres in the female prgeny were significantly higher than the titres in the male prgeny and much clser t the values f C57BL/6 than t thse f BALB/c. An analysis f the titres f the male prgeny revealed a significant difference (P < 0.01) between the prgeny f the tw crsses in that the titres f the C57BL/6 x BALB/c prgeny were higher than thse f the BALB/c x C57BL/6 prgeny. The female prgeny f bth crsses had cmparable titres (Table 1, Fig. 1). Table 1. HSV-induced 1FN titres in parental strains and F1 hybrids BALB/c C57BL/6 F1 BALB/c x C57BL/6 F1 C57BL/6 x BALB/c t & r A ~ t A ~ r A ~ 6`? 6` 9 6` ~ 6` Mean titre 2-15 1-96 3-39 3.08 2.99 2-20 2-95 2-38 Standard deviatin 0.14 0.17 0.18 0.28 0.49 0.19 0.43 0.29 Number f mice 13 18 13 18 10 24_ 17 9'~ Level f significance \ / \ / ~ / / ~ \ /7 between $ and 6' P < 0.005 P < 0.005 P < 0.00 P < 0.005 between FI ~. / between F1 6` P < 0.01 Dwnladed frm www.micrbilgyresearch.rg by
Interfern and herpes simplex virus 327 4, I I I I I I! ~ ~ 3 : @e g g g 2 :. ::. : g..... nr....... :... 2..... ~..... : 2. 888 ~-...~..-... g... ~ $II" -- ZlIS I~,INNbO I 1 I C57BL/6 I I I I I I BALB/c C57BL/6 BALB/c BALB/c F1 F1 BALB/c F1 C57BL/6 F1 C57BL/6 F2 C57BL/6 BALB/c Fig. 1. Genetic analysis f HSV-induced circulating IFN levels. Titres were measured in parental strains, F1, F2 and backcrss generatins. The pen symbls represent values btained frm female animals, the clsed symbls males. Table 2. HSV-induced IFN titres in F2 and backcrss generatins F1 (BALB/c x C57BL/6) F1 (BALB/c x C57BL/6) F2 (BALB/c x C57BL/6) C57BL/6 backcrss x BALB/c backcrss g g f Mean titre Standard deviatin Number f mice Level f significance between? and c~ 3.17 2.68 3-29 2-82 0.39 0.49 0-28 0"23 30 30 15 31 2.58 2-23 0-36 0.26 15 40 P < 0-001 P < 0-001 P < 0.001 F2 generatin: (BALB/c x C57BL/6)F1 x (BALB/c x C57BL/6)F1 The IFN titres f the F2 prgeny were als significantly different fr males and females. Again, the titres were higher in the female prgeny than in the male prgeny, and the difference between the mean values f bth was highly significant (P < 0.001). Furthermre, the values btained frm males were spread ver the whle range f BALB/c t C57BL/6 titres, whereas thse frm females were nly f intermediate and f C57BL/6 type (Table 2, Fig. 1). Dwnladed frm www.micrbilgyresearch.rg by
I I I I I i I I I I, i I I I I i I i! C57BL/6 BALB/c CXBD CXBE CXBG CXBH CXBI CXBJ CXBK HW94 Fig. 2. Genetic analysis f HSV-induced circulating IFN levels. Titres were measured in recmbinant inbred and cngenic lines. The pen symbls represent values btained frm female animals, the clsed symbls males. O0 N N O,..] Dwnladed frm www.micrbilgyresearch.rg by. c~ 0 ee e : :................ O 0 t 0 000 O0 O 00 O ~ go gee 0 0 go0 000 O00 000 OOO0 000 OO 0 O0 O0 O0 0O0O eg e ee ~ 0 OOe 831"... l...
Backcrss generatin (BALB/c x C57BL/6)F1 x C57BL/6 Interfern and herpes simplex virus 329 The female prgeny f this backcrss had IFN titres cmparable t thse f C57BL/6, whereas the titres f the male prgeny were intermediate, between C57BL/6 and BALB/c values. Again, the difference between male and female values was highly significant (P < 0-001) (Table 2, Fig. 1). (BALB/c x C57BL/6)F1 x BALB/c Abut half f the titres f the female prgeny fell within the range f the female BALB/c values whereas the rest were intermediate between female C57BL/6 and BALB/c. The same was true fr male prgeny when cmpared t male C57BL/6 and BALB/c mice (Table 2, Fig. 1). Recmbinant inbred strains and the cngenic B6-C-H-28c-lf-P (HW94) strain The strain distributin pattern (SDP) f HSV-induced IFN levels was further examined in seven RI lines derived frm C57BL/6 and BALB/c (Bailey, 1971). Fr all these strains, the level f IFN prductin was intermediate between C57BL/6 and BALB/c. Again the females had higher IFN titres than the males, and the difference was statistically significant fr six strains, the exceptin being the CXBD strain. The females f ne RI strain (CXBE) prduced values that came clse t the female C57BL/6 values but were nevertheless significantly lwer (P < 0-025). The values frm males f anther RI strain (CXBH) were clse t the male BALB/c values, but still significantly different (P < 0.05) (Table 3, Fig. 2). T lk fr pssible linkage with If-1 (DeMaeyer & DeMaeyer-Guignard, 1969), HSVinduced IFN prductin was measured in the cngenic B6-C-H-28 -If-11 (HW94) strain (DeMaeyer et al., 1975). In this strain als the IFN prductin f females was significantly higher than that f males; hwever, the levels were nt significantly different frm C57BL/6 levels (Table 3, Fig. 2). DISCUSSION A striking feature f the analysis f HSV-induced IFN levels in the present study is the significantly higher titres btained in female animals in all the crsses examined as well as in the parental, the RI and the cngenic strains. There are several indicatins that this difference is X- linked. The mst imprtant evidence is the utcme f the determinatin f IFN levels in the male prgeny f the F1 crsses in bth directins. The fact that the IFN levels f the male prgeny f the C57BL/6 x BALB/c crss are significantly higher than thse f the prgeny f the BALB/c x C57BL/6 crss clearly indicates an X-linked influence in this crss, since IFN levels are higher when the X-chrmsme riginates frm C57BL/6 than frm BALB/c mice. This is further brne ut by the IFN titres f female prgeny f the F1 generatin in bth directins, which are n average 3.7 times (fr C57BL/6 x BALB/c) and 6 times (fr BALB/c x C57BL/6) higher than the titres f the male prgeny, whereas the difference between average titre f males and females in the parental strains is nly 1-5 times (BALB/c) and 2 times (C57BL/6). This indicates that, when tw X-chrmsmes are present, the influence f the high prducer X-linked allele f C57BL/6 rigin is dminant ver all ther alleles influencing HSVinduced IFN levels. This is als suggested by the utcme f the F2 and F1 x C57BL/6 backcrss, in which the mean titre in the female prgeny is abut 3 times that f the male prgeny. Analysis f the distributin f the titres f the male and female F2 prgeny als reveals that several lci are invlved, since, if we were nly dealing with ne lcus, 25 ~ f the female F2 prgeny wuld be expected t have titres cmparable t BALB/c values. This wuld crrespnd t 7 r 8 (i.e. 25~) animals, whereas in the bserved result nly 1 (i.e. 3~) f the prgeny fell within the range f female BALB/c values. By the same reasning, ne wuld expect 75 ~ f the male F2 prgeny t have titres crrespnding t male BALB/c r F1 values, which are indistinguishable because f the cnsiderable verlap f the bserved values; in fact, nly 15 ut f 30, r 50~ fall within this range. The actual number f lci invlved, in additin t the X- linked lcus, cannt be accurately deduced frm the segregatin results, since t many Dwnladed frm www.micrbilgyresearch.rg by
K 3-00 0'34 10 2.44 0.16 7 P < 0.005 HW94 3"33 0.17 10 2-97 0'35 21 P < 0-005 ~0 N...] N Z 0 Dwnladed frm www.micrbilgyresearch.rg by Table 3. HSV-induced 1FN titres in RI strains and in the cngenic HW 94 strain Strain D E G H I J Mean titre 2.43 3.09 2.63 2.53 2.62 2.62 Standard deviatin 0.44 0.24 0.31 0.17 0-20 0.35 Number f mice 8 10 10 10 10 10 Mean titre 2.35 2.60 2.23 2.07 2-32 2.20 Standard deviatin 0.12 0.48 0.18 0.15 0.10 0.13 Number f mice 10 10 10 t 1 10 11 Level f significance between ~ and NS* P < 0.005 P < 0-005 P < 0-005 P < 0.005 P < 0-005 * Ns, Nt significant.
Interfern and herpes simplex virus 331 Table 4. Estimatin f the number f lci invlved in determining the levels f HSV-indueed IFN prductin frm segregatin in the F2 prgeny Female prgeny: Expected percentage f BALB/c-type prducers Observed percentage Number f lci: 1 2 3 25~ 6.2~ 1.5~ 3~ Male prgeny: Expected percentage f BALB/c- and Fl-type prducers Observed percentage Number f lci: 1 2 3 75~ 56~ 42~ 50~ assumptins cncerning the relative dminance f the different alleles wuld have t be made; the segregatin values suggest that tw r three lci are invlved (see Table 4). The results btained in the RI strains cnfirm that the trait under study is under multifactrial influence, since nne f the RI strains has a clear-cut parental phentype, and fr all strains the bserved values are intermediate between BALB/c and C57BL/6. The SDP f HSV-induced IFN levels in the RI strains furthermre shws n bvius crrespndence with any f the previusly bserved SDPs fr IFN inductin by NDV, muse mammary tumur virus r Sendai virus, thereby suggesting lack f clse linkage t any f the If-lci previusly described. Lack f linkage t If-1 is furthermre cnfirmed by the results btained in the B6-C-H-28c-If-11 strain, in which the HSV-induced IFN titres were f C57BL/6 type rather than f BALB/c type. The cnclusin f this study, therefre, is that the levels f early HSV-induced IFN prductin are influenced by several (pssibly tw) autsmal lci and ne X-linked lcus: nne f these is clsely linked t If-l, and prbably nt t any f the ther If-lci either. The latter pint, hwever, will have t be cnfirmed by further linkage studies, since, with the exceptin f If-l, n cngenic strains fr the ther If-lci are available. These results have several implicatins: lci influencing levels f IFN prductin in the muse have been previusly described fr ther IFN-inducing viruses, but n evidence fr X- linkage f these genes was bserved. Hwever, in the case f these ther viruses, early IFN prductin was nt studied in detail, since nly IFN levels at later times (8 t 12 h) were cmpared. Therefre, the pssibility that with sme f the ther inducers the level f early IFN inductin is als t sme extent under X-linked cntrl can nt be excluded. This pint certainly merits further investigatin, since a mre general influence f the X-chrmsme n the levels f IFN prductin with several viruses wuld have implicatins fr the physipathlgy f virus diseases in males and females. A Mendelian analysis perfrmed by Lpez (1980), using C57BL/6 and BALB/c mice as parental strains, prvided evidence that primary resistance f C57BL/6 mice t HSV-induced encephalitis is mediated by tw independently segregating lci; n evidence fr X-linkage was reprted in the analysis by Lpez. Hwever, tw recent reprts have prvided evidence fr an X- linked lcus r lci influencing resistance f mice t HSV-2-induced hepatitis (Mgensen, 1977) and HSV-2 infectin (D. Armerding, persnal cmmunicatin) and we believe that these bservatins are directly relevant t the results reprted here. Several reprts in the literature prvide evidence fr the existence f X-linked genes in man influencing susceptibility t Epstein-Barr virus (EBV), which belngs t the herpesvirus grup. Males affected with the X-linked lymphprliferative syndrme are uncmmnly susceptible t infectius mnnuclesis, and this disease ften has a fatal curse in such patients (Purtil, 1980). Interestingly enugh, these males frequently have deficient natural killer cell activity (Sullivan et al., 1980). In additin, naspharyngeal carcinma, in which EBV has been implicated as a causative agent, ccurs mainly in genetically predispsed males (fr review, see Purtil, 1981). In view f the highly cnserved nature f the X-chrmsme, we believe that the pssibility that herpesvirus-induced IFN prductin is X-linked als in man merits full investigatin. REFERENCES BAILEY, D. W. (1971). Recmbinant-inbred strains. An aid t finding identity, linkage and functin f histcmpatibility and ther genes. Transplantatin 11, 325 327. Dwnladed frm www.micrbilgyresearch.rg by
332 R. ZAWATZKY AND OTHERS DEMAEYER, E. & DEMAEYER-GUIGNARD, L (1969). Gene with quantitative effect n circulating interfern induced by Newcastle disease virus. Jurnal f Virlgy 3, 506-512. DEMAEYER, E. & DEMAEYER-GUIGNARD, L (1979). Cnsideratins n muse genes influencing interfern prductin and actin. In Interfern 1979, pp. 75-100. Edited by I. Gresser. New Yrk & Lndn: Academic Press. DEMAEYER, E., DEMAEYER-GUIGNARD, J. & BAILEY, D. W. (1975). Effect f muse gentype n interfern prductin. Lines cngenic at the If-1 lcus. Immungenetics 1, 438-455. DIXON, w. J. & MASSEY, F. J. (1969). Intrductin t Statistical Analysis, 3rd edn., p. 638. New Yrk: McGraw-Hill. GRAHAM, F. L., VELHUISEN, G. & WILKIE, N. M. (1973). Infectius herpesvirus DNA. Nature New Bilgy 245, 265-266. HOLLANDER, M. & WOLFE, D. (1973). Nnparametrical Statistical Methds, p. 68. New Yrk: Jhn Wiley & Sns. K1RCHNER, H., KOCHEN, M., HIRT, H. i. & MUNK, K. (1978). Immunlgical studies f HSV infectin f resistant and susceptible inbred strains f mice. Zeitschrift fur Immuniti~tsfrschung 154, 147-154. LOPEZ, C. (1980). Resistance t HSV-1 in the muse is gverned by tw majr independently segregating nn H-2 lci. Immungenetics 11, 87-92. MOGENSEN, S. C. (1977) Genetics f macrphage-cntrlled resistance t hepatitis induced by herpes simplex virus type 2 in mice. Infectin and Immunity 17, 268-273. MORGAN, J. F., MORTON, H. F. & PARKER, R. C. (1950). Nutritin f animal cells in tissue culture. I. Initial studies n a synthetic medium. Prceedings f the Sciety fr Experimental Bilgy and Medicine 73, 1-8. PURTILO, D. (1980). Epstein-Barr-virus induced ncgenesis in immune-deficient individuals. Lancet i, 300-303. PURTILO, O. (1981). Immune deficiency predispsing t Epstein-Barr-virns induced lymphprliferative diseases: the X-linked lymphprliferative syndrme as a mdel. Advances in Cancer Research 34, 279-312. SULLIVAN, J., BYRON, K., BREWSTER, E. & PURTILO, D. (1980). Deficient natural killer cell activity in X-linked lymphprliferative syndrme. Science 210, 543-545. ZAWATZKY, R., HILFENHAUS, J., MARCUCCI, F. & KIRCHNER, H. (1981). Experimental infectin f inbred mice with herpes simplex virus type 1. I. Investigatin f humral and cellular immunity and f interfern inductin. Jurnal f General Virlgy 53, 31-38. (Received 2 June 1982) Dwnladed frm www.micrbilgyresearch.rg by