Macrophage activity in rabies virus infection of genetically selected high and low antibody responder lines of mice

@ INSTtTUTPASTEUR/ELSEVIER Paris 1990

Res. ViroL

I990, 141, 57-67

~:~ :;ROPHAGE ACTIVITY IN RABIES VIRUS INFECTION OF '~ZNETICALLY SELECTED HIGH A N D LOW ANTIBODY RESPONDER LINES OF MICE C.A. Consales (t), R.Z. Mendonea (~), M.A. Lucchiari (~), R.C. Vassao (z) and C.A. Peveira (:) (*) (i:~

.:~:lo Butantan, Laboratorio de Imunologia Viral, Av, Viga4 Brasi[ 150~, CP 65, 05504 Sao Pauto, and (2) htstituto Pasteur, Sao Paug,:~ (Brazil)

SUMMARY After infection with the Pasteur strain o2 ixed rabies virus, the onset of disease, mortality, interferon (tFN) s?~thesis and interaction of the virus with macrophages were investigated in b{gh (H0 and low (L-0 antibody responder Iines of mice. The HI mice were shovm to be more resistant than the Lz mice, and resistance was age-dependent, ;i~n.¢enfice from both mouse Iines were fully susceptible up to 2 weeks of age. iY?-.~sbmthesis studies of the serum indicated that, after rabies infection, Ht mice produced a slightly higher amoun~ of IFN, which was determined m be predominantly IFN-gamma. In the brains of LI mice, only IFN-alpha/beta was found, in contrast to the mixture of IFN-atpha/beta and IFN-gamma observed in the brains of HI mice. Although macrophages from the two mouse Iines expressed the same degree of ex:~rinsic activity, their intrinsic activities were quite different; the L~ mice showed a greeter ability to uptake and process the virus or ingest C3 (IgM) sheep red blood cells. The present findings atzribute the higher antibody response and IFN-gamma s3mthesis observed in H~ mice during rabies infection to slower processing of the rabies antigen in their macrophages, thus conferring upon them a greater ability to present it to the immune system, leading to a higher degree of resistance to rabies infection.

Submitted September 7.. ~989, accelptedNe.vemb--r.30, 19-C9. (*}Cor,:'es.,:~ndencezo Dr Car~osA, Pereira,

58

C.A. C O N S A L E S E T A L .

KEY-WORDS" Rabies, Macrophage, lmmunogenetics, Interferon; Antibodies, High and low responder mice.

INTRODUCTION

After experimental rabies infection, the virus initially remains near the site of inoculation during which time virus replication m a y occur. Unless neutralized by specific or non-specific defence mechanisms, the virus then enters the peripheral nerves and centripetally reaches the central nervous system, where it can be found at high concentrations with minimal neuropathological findings (Emerson, 1985). Different mouse strains show different patterns of resistance to rabies infection, and several mechanisms such as humoral or cellular immune responses and antivira! effects induced by IFN are suggested to be involved in resistance (Lodmell and Chesebro, 1984; LodmelI and Ewa!t, 1985; Pereira et aL, I982). High (H0- and low (L0-responder mice from genetic selection I were assayed for antibody responsiveness to heterologous erythrocges (Biozzi et aL, 1979). The difference between H! and L I lines is not oniy restricted to responses against the selection antigens (heterologous erythrocytes) but to accumulated high er low alleles that were found to regulate the amount of antibodies against a wide range of unrelated immunogens (Biozzi et aL, t979). Besides this "multispecific effect" of selection, other immunological parameters have been modified in H~ and L~ mice; the most important concerns macrophage activity: the bactericidal and catabolic activities of macrophages are higher in the LI than in the H~ responder line (Biozzi et aL, 1979; Wiener and Bandieri, 1974). H t and LI lines from distinct genetic selections have been successfully emNoyed to characterize the main immunological factors actipg in innate or acquired resistance to several pathogens (reviewed in Biozzi et aL, 1984) including rabies virus (Nilsson et al., 1979). Numerous observations in different host-virus systems have suggested that virus-macrophage interactions may determine the outcome of viral disease ~logensen, 1979; Pereira et al., 1984b ; Turner and Ballard, 1976) and that the degree of macrophage activation may be an essential step in this process ~Iogensen, 1979; Pereira el aL, I985).

FCS Hr i.c. i.m. Lp. IFN L~ LPS

= = = = = = = =

foetal c a l f ser'am. h i g h r e s p o n d e r (line o f mice)~ intracerebral(ly). intramusculax(ly).

intraperitoneal(b,). interferon. low r e s p o n s e r (line o f mice). lipopolysaccharide.

MICLD¢~ m.o.i. PE PEC PV SRBC TCID..~

= = = = = = =

mom.e ~ r a c e r e b r a ! 50 ~e l~th~ dose. multipiici~T o f irffe~tiom peritoneal exudate. p e ~ t o n e a l exudate cetl. P a s t e u r strain o f fixed rabies virus. ~heep r e d b l c ~ d celt. t~ssue-cultureqnfeeti~'e d o s e 50 % .

RABIES IN HIGH AND LOW RESPONDER t~ICE

59

The present study was u n d e r t a k e n to obtain data concerning the role o f m a c r o p h a g e s and I F N during experimental infection with rabies virus. Results presented here favour the view that m a e r o p h a g e s a n d their activation by I F N - g a m m a play an essential role in the first phase o f rabies infection.

MATERIALS AND METHODS Virus. A Pasteur strain (PV) of fixed rabies virus adapted to BHK21 ceil culture (kindly supplied by Prof. P. Sureau, Institut Pasteur, Paris) with a titre of 104-8 mouse intracerebral 50 % lethal doses (MICLD~0/0.03 ml), as calculated by the method of Reed and Muench, 1938, was used in ~II experiments. Mice.

H~ and Lt mice c,riginated from the Instituto Biologico, Sao Paulo and were bred in our mouse colony and used in the experiments at the ind'cated ages. In the virus titration tests (Koprowski, 1973), Swiss albino mice from the outbred coto~ly of the Instituto Butantan were used. Virus infection.

H I and L i mice at the indicated ages were intramuscularly (Lm.) i~._fected with .105 MICLDs0/0.3 ml and observed for 30 days. During this time, cl~rdca| symptoms and mortality were observed daily and recorded. IFN assay. A cytopathic-effect reduction test technique described in detail elsewhere (Pereira etaL, 1984a) was used for the IFN titre determination. Briefly, monolayers of L929 cells in 96-well microtitre plates were incubated for 18 h with 2-fold dilutions of sera or supernatants of brain homogenates. Sera and brain suDernatants were then removed and monolayers were infected with t00 t.~ssue-culture-infecfive doses 50 % (TCID_~) of encephalomyocarditis virus. Unadsorbed virus was removed 2 h later by washing the monolayers, and fresh minimal essential medium (MEM) supplemented with 10 % foetal calf serum (FCS) was added. Microtitre plates were incubated for 72 h and the ~FN tkre (in units/ml) was expressed a~ ~he reciprocal dilution of the serum or brain supernatant able to inhibit 50 70 of the virus replication. For characterization of IFN-alpha/beta or -gamma in the samples, poiycionai antibodies to mouse IFN alpha:~eta and monoclonal antibodies to mouse recombinant IF_ :~-garrtma with activity of 2 × 103 and 2 × l0 s neutralizing units per mg, r~pecfively, were airways used. The antibodies showed no cross-reactivity, and controls far !FN characterization included ~aterwal and well-known preparations of IFN-alpha/beta, ~FN-gamma and a mkxtme of both to assure the assay specificity and sens~tNity. Phagocytosis assay.

Peritoneal macrophages from H~ and L~ ~dce were collected by peritoneal lavage with RPMI-1640 medium with 10 % FCS, cultured in coverslips in 24-well plates

60

C.A. CONSALES ET AL.

and washed 3 times after 1-h incubation at 37°C with 5 % CO2. Cultures were utilized after 24 h of incubation. The technique used for the phagocytosis assay has already been described (Pereira et at., 1987). Briefly, sheep red blood cells (SRBC) stored in Alsever's solution at a concentration of 5 % were incubated with rabbit lgM anti-SRBC for 15 rain. The igM~SRBC were then incubated with a 1/20 dilution of C5-deficient serum from AKR mice yielding C3 (IgM) SRBC. The preparation was washed and resuspended in RPMI-1640. Macrophages activated for 18 h with the indicated concentrations of iipopolysaccharide (LPS) from E. coli O111 :B4 (Difco Laboratories) were incubated with C3 (IgM) SRBC preparation at 37°C for 60 rain, treated with 0.14 M solution of NH4CI at 37°C for 2 min, fixed in 2 °7o glutaraldehyde; the coverslips were washed, stained by the Giemsa method and mounted. Results are expressed by the index of ingestion, which is the percentage of cells with ingested SRBC multiplied by the average number of SRBC ingested per cell. At least 200 cells were countec for the percentage of ingestion and 50 ceils for the average number of SRBC ingezted.

Intrinsic and extrinsic macrophage activities. In order to assess intrinsic activky, or the ability of macrophages to uptake and process the virus, peritoneal exudalz (PE) and peritoneal exudate cells (PEC) were collected from the peritoneal cavity of mice previously infected intraperitoneally (i.p.) with 105 MICLDs0 of PV. Each sample of PE and PEC was then intracerebrally (i.c.) inoculated into 10 newborn Swiss mice" mortality was observed during the following 30 days and the per cents recorded. For extrinsic activity, or the ability of macrophages to inhibit virus growth in BHK21 cells, cultures of BHK21 cells on cover slips were infected with I m.o.L (multiplicity of infection) of PV for 3 h at 33°C; then, 104 effector PI~C were added, and the cultures incubated for 96 h, fixed for 30 rain with cold acetone, washed tw-ice with phosphate-buffered saline (PBS) pH 7.2 and labelled by a 30-min incubation with fluoresccin isothiocyanate-conjugated monoclonal antibodies against the nucleocapsid of rabies virus (Institut Pasteur, Paris) diluted to 1/100. They were taea washed twice with PBS pH 7.2, mounted with glycerine and observed with an epifluorescence "Reichert" microscope. Per cent positive cells was obtained by counting more than 100 cells and recording those showing immunofluorescent labelling. RESULTS

Mortality of mice after rabies virus infection. In figure 1, we show the onset of disease and mortality of adult HI and L~ mice after i.m. infection with l0 s MICLDs0 of rabies virus. The HI mice showed a much higher resistance to the infection. Although, in animals developing rabies, the period of incubation was the same, with the appearance of disease at day 6 or 7, H~ mice were able to resist for a longer time after the onset of disease. In table I, the study of age dependence in resistance showed that up to 14 days of age, both mouse fines were fully susceptible to the rabies virus infection, and at adult age (30 or 60 days), the HI mice developed a higher degree of resistance than LI mice. Mice from both mouse lines which developed the disease showed the same clinical symptoms of rabies after the sixth day of infection, characterized by tremors when held in the air by the tail with forceps, lack of coordination of hind legs and paralysis.

RABIES

HI MICE

3o!

IN HIGH AND

LOW

15

0

5

RESPONDER

MICE

6t

kP,l;tt5 ' 11'

8

14

DAYS

L'3°ll iNilll ! 15

o

~'i

5~

El

!i

11

14

DAYS FrG. 1. ~ 2klortality and disease of 30-day oM mice after Lm. injection with tGJ MICLD_,o o f PV. [] = mice without s3n-nptoms; N = mice with rabies s;~mptoms; tl = dead mice. Tgirty mice were injected and observed for 30 days.

TABLE I. ~

A g e - d e p e n d e n t m o r t a l i t y o f H I a n d L] m i c e a f t e r i n f e c t i o n ~ i t h P V . Mortality Age (days)

N

Hr

14 30 60

30/30 3/30 0/30

100 10 0

Li

14 30

30/30 19:'30

60

15/30

I00 63.3 50

Mice

%

Mice were infected i.m. with l0 s MiCLD~ of PV and ooserxed for 30 days. Results indicate the mean values of 3 different experiments which showed a slight varia~2om

C.A. C G N S A L E S E T A L .

62

Synthesis o f I F N in mice infected with rabies virus. In table II we show the synthesis o f I F N - a l p h a / b e t a and _~FN-garnma in sera and brains o f Hr a n d LI mice at d i f f e r e n t times a f t e r ~ffection with PV. Hi mice were s h o w n to p r o d u c e slightly higher titres o f I F N - g a m m a in s e r u m at 5 days after infection w h e n c o m p a r e d to t h o s e produce,~ in LI mice. N o I F N - g a m m a was d e t e c t e d in the brai,.s o f L~ mice. h- b e t h m o u s e line~, the p e a k o f I F N synttlesis was always observed to a p p e a r !ater in the b r a i n t h a n in the serum.

TABLE II, - - Synthesis of IFN in ihe serum and brain of Ii I and L I mice infected with PV. IFN titres (units iFN/ml or g) at N days after infection Mice

Tissue

HI

Serum alpha/beta ~arrana Brain alpha/beta gamma Serum alpha/beta gamma Brain alpha/beta

LI

IFN type

4

5

6

7

400 0 0 0 200 0 0

400 1,200 650 0 400 400 1,300

0 400 1,300 1,300 0 400 2,600

0 1(,3 1,300 1,300 0 100 2,600

Mice wereinfectedi.m. with 105 MICLD~0 of PV, sacrificed,and serumand brains collected.IFN titre resultsare the meanvaluesof at !east5 micein 3 differentexperimentswhichshoweda slightvariation.

TABLE III. - - Ingestion of C3 flgM) SRBC by macrophages from HI and L~ mice, Ingestion of C3 (IgM) SRBC Macrophages HI

LPS (~tg/ml) -*10 20

LI 10 20

%

No. per ceil

Index

21 80 78 75 02 96

1.76 4.03 4.12 2. I6 4.32 4.80

36.9 322.4 321.3 162.0 397.4 460.8

Peritoneal macrophagesfrom H~aa~ L~mice werecultivatedon coverslips and activatedwith the indicatedamountof LPS. Iag~-tioni~-dexi~the percentageof macrophageswithingestedSRBCmultiplied by the averagenumber ef SRBC ingest~a~er maerophage.Data express at least 200 cells counted for percent of ingestion, and 50 ceils for averxg¢number of SRBC ingested. Results indicatemean values from 3 differentexperimentswhh~hshowed a slight variation.

tLABIES I N H I G H A N D L O W R E S P O N D E R M I C E

63

Ability of macrophages to ingest C3 (lgM) SRBC. As indicated in table III, a high percent o f m a c r o p h a g e s f r o m L I mice were capable o f t a k i n g u p C3 (IgM) S R B C w i t h o u t stimulation. A f t e r L P S stimulation, t h e m a c r o p h a g e s f r o m b o t h m o u s e lines ~.kowed t h e s a m e degree o f ingestion enhancement.

Intrinsic and extrinsic activity of macrophages. T h e results o f t h e : t u d y o f t h e intrinsic activity o f m a c r o p h a g e s are showT~ in table IV, a n d indicate t h a t m a c r o p h a g e s f r o m L I mice were capable o f u p t a k i n g , p r o c e s s i n g a n d clearing t h e i n o c u l a t e d rabies virus m u c h faster t h a n m a c r o p h a g e s f r o m t h e HI mice. Five h o u r s after i n o c u l a t i o n , n o virus c o u l d be f o u n d in P E o r P E C o f LI mice. H o w e v e r , even 5 a n d 8 h after inoculation, respectively, we f o u n d virus in the P E C a n d P E o f HI mice. Macrophages f r o m t h e t w o m o u s e lines were c a p a b l e o f expressing t h e s a m e degree o f extrit_,~ic activity, t h e r e b y i n h i b i t i n g virus g r o w t h in B H K 2 I cells (table V).

TABLE IV. - - Intrinsic activiW of macrophages from H I and LI mice. % Mortality (indicating the presence of virus) at hours after infection Mice HI Li

Material

1

5

8

t8

, PE PEC PE PEC

100 I00 90 80

50 5 0 0

20 0 0 0

0 0 0 0

Micewerei.p. infectedwith l0 s MICLD~0of PV. PE and PEC wereobtainedlater and i.e. inocalated into I0 newbornSwissmice.Resultsare the mean valuesof 3 differentexperimentswhichsho~,eda slight variation.

TABLE V,

~

Extrinsic activity of macrophages from HI and L~ mice. % Immunofluorcscence Control I-it h

78.0 5.3 5.9

BI-FK2-I cellmonCayerson cove~stipswereinfected~ith I m.o.i, of PV for 3 h at 3MC, at whichtime 1~ PEC were added. After 96-h incubation, cells were hilled by imm~mofluoresomce,as described in "biatcrials and Methods".Percentagesof pod~veedls wererecorded,Controlindicatesresultin the of macrophagcs.Resultsare racan vaIuesfrom 3 differem experimentswhich showed a dight variation,

64

C.A. C O N S A L E S E T A L .

DISCUSSION It has been recognized that host genetic factors influence murine resistance to various viral infections (Biozzi et al., 1979; Lodmelt and Chesebro, 1984; Lodmei1 and Ewalt, 1985; Mogensen, t979; Nilsson et al., 1979; Pereira et al., 1984b; Wiener and Bandieri, I974). The genetic factors affecting the phenotypie expression of resistance or susceptibility to infection, depending on the model of study, may be visualized under either monogenic or polygenic control, operating at both specific and non-specific tevels. Macrophages are considered as key cells during viral infections, playing a role in non-specific as well as specific immune mechanisms, and may determine the resistance or susceptibility of the animal to a given virus (Mogensen, 1979; Pereira et al., 1984b; Pereira et al., 1987; Turner and Ballard, 1976). The HI and L I lines of mice, which were selected :['or maximal and minimal humoral antibody formation, and the genes which accumulated in these lines were found to regulate the amount of antibody produced against a wide range of unrelated antigens, including rabies virus antigens (Ni!sson et al., 1979). Macrophages from LI mice were shown to be more active than H I maerophages; as a consequence, antigen attached to the surface of LI macrophages could be rendered less immunogenic, explaining the minimal humoraI antibody formation (Biozzi et at., 1979; Wiener and t~andieri, 1974). After infection with PV, H i mice showed a much higher degree of resistance (fig. 1), which was associated with the higher !evels of antibodies a~,m,~ ~,~,,,;, detected in these animals when immunized with ultravioletinactivated raNes virus (data not shown). These data confirm previous observations concerning the positive correlation between the resistance of high and low mice from selection III and IV wkh specific antibody formation (Nilsson et aL, I979). The obselwation that the resistance of Hi mice is age-dependent (table I) agrees with the hypothesis of the main role of the immune response through specific antibody formation, wh;,ch reaches high levels with the maturity of the immune system. The incubation period of disease was found to be the same in both mouse lines; however, H I mice resisted for a longer time after the onset of disease (fig. 1) indicating that H I mice not only resist the onset of disease but also slow down viral progression after the appearance of the first symptoms. The study of the i:~trinsic activity of macrophages from H I and L, showed that macrophages from LI mice were able to uptake, process and clear the rabies ~Arv.smuch faster than macrophages from Hi mice (table YV~).These data are in agreement ~s4th the experimen~ showing that phagoc3~os~s of C3 (IgM) SRBC occurs in macrophages from L t mice even without previous stimulation by LPS (table HI). L~ macrophages were clearly more active than HI macrophages in terms of C3 (igM) SRBC phagocytosis as well as rabies virus uptake and processing.

R A B I E S I N H I G H A N D L O W R E S P O N D E R ~,flCE

65

Mechanisms that may be involved in the resistance or susceptibility shown by these mice include macrophage activation and the antiviral effect mediated by IFN-gamma. In rabies-infected HI mice, we detected higher titres of IFN-gamma compared to those of LI mice (table II), which can be explained by the greater ability of HI macrophages to present antigen to the immune system., leading to a high level of IFN-gamma production. Other than the possible role of IFN-gamma in the induction of the antivirat effect, by inhibiting vira! replication in HI mice and leading to higher resistance, we speculate that the well-known ability of IFN-gamma to activate macrophages may play a role in potentiating the already enhanced activity of L~ macrophages. Such a mechanism would further increase the hyperacti_vity of L~ macrophages by the IFN-gamma produced, leaaing to a state of susceptibility due to deficient antigen presentation. These findings are in accordance with others in the literature (Biozzi et aL, 1979; Nilsson et at., 1979; Turner and Ballard, 1976; Wiener and Bandieri, 1974) and may explain the poor immunogenic ability of L! macrophages, which can be further potentiated by the production of tFNgamma and the presention of the rabies anr'.'-gen to immunocompetent ceiis, so induciv.g a lower level of rabies antibody production, and possibly IFNgamma, after infection. On the other hand, no differences were observed in extrinsic activity in macrophages of the two mouse lines (table V). They were equally capable of inhibiting virus growth in BHK21 cells. Since extrinsic actMty seems to be dependent on sotubIe factors released from macrophages, such as IFNalpha/beta, produced in comparable amounts in both mouse lines (table I!), and intrinsic activity is dependent on hyperactivit3j of the macrophage membrane, augmented endocytosis and/or a well-developed iysosoiiial system, it is not surprising that macrophages from HI and LI mouse lines behave differently in their intrinsic actMty but not in their extrinsic actMty. In conclusion, the data presented here show that the differences in the resistance levet of H~ and L1 mice to rabies infection are ~n direct correlation with the intrinsic activities of their macrophages, which possibly influence the development of an immune response capable of inhibiting virus particles and determining the outcome of the disease..~ for rabies in.fection, the seIectcd high and low antibody responder lines provide useful biological materiat for the study of mechanisms involved in resistance to pathogens. RI~SUMg ACTION DES MACROPI-L%OESD.M'ZSL'INFECTION RAE,"QUECHEZ DE5 ~OURIS SI~LECTIONF-ESGI~:,'£1~TIQUEMENTPOUR UNE BONNE ET .k,/~.UVA~E REPON~E .ANT~C,ORPS

Apr6s infection par ta souche Pasteur (PV) du virus rabique fix4 de !ign6es des souris bonnes et mauvaises r6pondeuses eta anticorps (H~ et kt), nous av~.~nsetudi6 l'apparition de la maladie, Ia mortalit6, la synth~se d'interferon .(IFN) et t'interac.tion du virus avec ies macrophages. Les souris H I se son~: rnontr6es plus rOsistantes

66

C.A. C O N S A L E S E T A L .

que les souris Lr, et cette r&istance est fonction de l'fige, puisque les souris des det~x lign&s sont totalement sensibles jusqu"~ l'~tgede deux semaines. L'&ude de la synth~se d'IFN dans le s&um a montr6 quetes souris H 1 ont produit, a::r~s t'infection raNque, une quantit6 d'IFN plus 6Iev&, qui a 6t6 d6terminfe comme &ant err majeure pattie de l'IFN-gamma. Dans le c~2rveau des souris Li on n'a trotiv6 que de I'IFNalpha,b&a, contrairement au m61anged'IFN-aIpha,b&a et IFN-gamma trouv6 darts te cerveau des souris H~. Bien que los macrophages des deux lign&s de scuris aient exprim~ le marne degr6 d'activit6 extrins~que, t'activit6 intrins,~que de ces m~mes macrop~ages est tr~s diffdrente, ceux des souris Li ayant un pouvoir plus importarlt de captation et de d6gradation du virus rabique ou d'ingestion des C3 (IgM)/globralos rouges. Ces r~sultats peuvent expl~quer l'augmentation de la r6ponse antieorps et cello de la synth~se d'IFN chez les souris H~ pendant l'infection rabique, augmentations d6pendentes d'une m6tabolisation pii~s !ente de l'antig~ne rabique par los ~lacrophages, ce qui rend possible une plus grande capacit6 de pr6sentation de l'antig~ne au systbme immunitaire, et, par cons6quent, un accroissement de ta r6sistance l'infection rabique. Mo:ts-cr_~s : Rage, Macrophage, Interferon, Immunog6n6tique; Anticorps, Souris bonnes et mauvaises r6pondeuses.

ACKNOWLEDGEMENTS This work was supported by grants from the Instituto Butantan, FAPESP (86/2979-5) and CNPq (400667/87L We thank Dr O.A. Sant;Anna and Dr O.A.C. Pereira for helpt)~i comments.

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