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J. gen. Virol. (1982), 63, 315 323. Printed in Great Britain 315 Key words: herpes simplex virus/virulence and pathogenesis/interferon/immunity Experimental Infection of Inbred Mice with Herpes Simplex Virus. V. Investigations with a Virus Strain Non-lethal after Peripheral Infection By G. K U M E L , * H. K I R C H N E R , R. Z A W A T Z K Y , H. E N G L E R , C. H. S C H R O D E R AND H. C. K A E R N E R Institute for Virus Research, German Cancer Research Centre, Heidelberg, Federal Republic of Germany (Accepted 7 July 1982) SUMMARY The herpes simplex virus type I(HSV-1) strain A N G , unlike the majority of HSV-1 isolates, does not cause lethal encephalitis in various inbred mouse strains, when applied in doses up to 2 x 107 plaque-forming units intraperitoneally, intravenously, intravaginally, orally, or via the foot pads. We studied and compared the progress of infection by this non-lethal strain and by a lethal HSV-1 strain, and the components of the host defence mechanism involved. If injected intracerebrally, HSV-1 A N G replicated efficiently in mouse brain cells and led to encephalitis. Upon systemic or peripheral infection, it replicated in several mouse organs with a virulence similar to lethal HSV-1 isolates. It was clear that transport of HSV-1 A N G to the central nervous system (CNS) or replication in CNS tissue is efficiently restricted after peripheral infection. Conceivably, infection with lethal HSV-1 strains proceeds in two distinct steps, virus replication at the site of infection and in the spleen and, secondly, transport to CNS tissue and propagation in CNS cells. This second step is apparently blocked in infections of DBA/2 mice by HSV-1 A N G and can thus be studied separately. The blocking mechanism was not a function of interferon induction or sensitivity, nor was it due to an enhanced N K cell activation. Experiments with silica-treated mice, and with homozygous nude mice, which lacked T-lymphocytes, suggested that the observed restriction in virus transfer is independent of T-cell and macrophage functions. Yet, newborn mice were fully susceptible to intraperitoneal infection with HSV-1 A N G , suggesting that age-dependent defence mechanisms, the nature of which needs to be further examined, are of relevance in the restriction of peripheral infection by HSV-1 A N G in adult mice. INTRODUCTION Herpes simplex virus type 1 (HSV-1) is a neurotropic human virus of considerable clinical importance, particularly in immunoincompetent or immunosuppressed individuals (Nahmias et al., 1981). With the aim of better understanding the pathogenesis of HSV and the host defence mechanisms interacting with the infection, model systems have been established in a number of laboratories (including our own) using HSV infection of mice (e.g. Andervont, 1927; Zisman et al., 1970; Stevens & Cook, 1971 ; Lopez, 1975; Kirchner et al., 1978). It is known that different strains of HSV-1, even primary clinical isolates, differ considerably in their lethality for mice (Hill et aL, 1975). Thus genetic differences between HSV-1 virus strains may be responsible for differences in pathogenicity. Most studies concerned with the mechanism of pathogenicity of HSV in mice have been performed using virus strains that cause lethal encephalitis in susceptible inbred mice at moderate virus doses. It is of interest to compare the spread of virus infection following peripheral infection with HSV strains of different pathogenicity patterns. Consequently, this study is concerned with the characterization of the infection of inbred mice with HSV-I A N G , a non-lethal HSV strain. 0022-1317/82/0000-5175 $02.00 © 1982 SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 08:27:14 316 G. KUMEL AND OTHERS In the past few years, the molecular genetics o f HSV-I A N G have been extensively studied in our laboratories (Schr6der et al., 1975/76; K a e r n e r et al., 1981; Kiimel et al., 1981, 1982). Recently, we have found that HSV-I A N G is non-lethal for mice after intraperitoneal (i.p.) infection (Schr6der et al., 1981). This was in marked contrast to HSV-1 W A L which has been commonly used in studies in our laboratory and which kills certain strains of inbred mice after i.p. injection of low doses of infectious particles (Kirchner et al., 1978). In this study, we analyse the spread of HSV-1 W A L and HSV-1 A N G in different tissues at various times after i.p. inoculation and attempt to define the host mechanisms which mediate the observed difference in behaviour of the two virus isolates. METHODS Cells and virus. HSV-I strains were routinely passaged at low multiplicity on African green monkey kidney cells (RC-37 Rita; Italdiagnostics, Rome, Italy) as described earlier (Schr6der et al., 1975/76). Cells were grown in Eagle's minimal essential medium (MEM) containing 7~ foetal calf serum. Plaque-forming virus was assayed as described by Russel (1962). Origin and characterization of the HSV-I strains ANG and WAL have been described earlier (Darai & Munk, 1976; Schr6der et al., 1975/76, 1981). Mice. The animal model used has been described in detail (Zawatzky et al., 1981). Inbred mouse strains, C57BL/6 nu/nu mice and their heterozygous litter-mates were purchased from GI. Bomholtgard Ltd. (Ry, Denmark) and from Deutsche Gesellschaft fiir Versuchstierkunde (Hannover, Germany). In all experiments with adult mice, 8-week-old animals were used. Newborns were bred in our department. Organ preparation. For the preparation of the spinal cord, the spine was freed from adherent muscle tissue and opened carefully, removing the dorsal part of the vertebrae until the spinal cord could be removed as a whole. Other organs tested included brain, liver, spleen and blood. Dissected organs were disintegrated by ultrasonic treatment and by freezing and thawing before titration of infectious virus. lmmunosuppression, lmmunosuppression by cyclophosphamide was done as described by Rager-Zisman & Allison (1976). A dose of 150 mg/kg of Endoxan (Asta-Werke AG, Bielefeld, Germany) was applied simultaneously with the virus. Suppression of macrophage functions was achieved by the injection of 50 mg silica suspended in isotonic NaC1 according to Zisman et al. (1970). Mouse interferon. Mouse interferon was induced with Newcastle disease virus (NDV) in C-243 cells, by an adaptation of the procedure developed by Cachard & DeMaeyer-Guignard (l 981). Briefly, monolayers of C-243 ceils in Petri dishes were incubated for 48 h in the presence of butyric acid (1 raM). After washing, monolayers were infected with N DV (input m.o.i, of 2) and virus was allowed to adsorb for 2 h. The cells were then washed twice to remove non-attached virus and incubated for 24 h in serum-free medium (MEM) supplemented with 3 mMtheophylline. Supernatants were harvested, centrifuged and stored at pH 2 for 5 to 7 days. This treatment proved to be sufficient for total inactivation of NDV. Control preparations were performed identically, but without adding NDV. The interferon preparation contained the three major protein species (35K, 28K and 22K), as analysed by polyacrylamide gel electrophoresis. The antiviral activity of this interferon preparation was 5 x l0 s IU/ml, hence having a specific activity of 1.6 x 107 IU/mg protein. Test of natural killer (NK) cellactivity and interferon induction. For testing NK cell activity a 4 h s 1Cr release assay was used with mycoplasma-free YAC-I lymphoma cells as targets (Engler et al., 1981). Interferon titrations were performed exactly as described by Beck et al. (1980) using a one-step assay, L-cells and vesicular stomatitis virus. RESULTS R e s i s t a n c e o f various m o u s e strains to H S V - 1 ANG We have shown previously that HSV-1 A N G does not cause lethal encephalitis in D B A / 2 and C57BL/6 mice on intraperitoneal (i.p.) infection (Schrrder e t al., 1981). In the present study, we have tested these and other inbred strains of mice including A / J , Balb/c and C3H, the sensitivities of which to pathogenic HSV-1 strains, including HSV-1 W A L , have been shown previously (Kirchner e t al., 1978). The results indicated that HSV-1 A N G was non-pathogenic in all cases, even at doses as high as 2 × 107 p.f.u./mouse. It has been shown (Kirchner et al., 1978) that D B A / 2 is highly susceptible to HSV-I W A L , and it was therefore chosen for most of the experiments with HSV-1 A N G described here. We have confirmed that less than 10 2 infectious particles of HSV-1 W A L are required to cause lethal encephalitis after i.p. infection in D B A / 2 mice. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 08:27:14 2 HS V strains differing in pathogenicity for mice 317 100 60 5x 103 < 20 2 10~ 5 I 6 8 4 Time post-infection (days) 5 1°"5 10 Fig. 1. Intracerebral infectionof DBA/2 mice with HSV-1ANG. Groups of at least 10 4- to 6-weeks-old animals were individually infected i.c. with the indicated numbers of p.f.u. HSV-1 ANG suspended in 0-02 ml buffer. Outcome of the infection by HSV-1 ANG when using different inoculation routes For comparison, we tested the pathogenicity of HSV-1 A N G following (i) systemic routes, (ii) peripheral routes and (iii) the intracerebral (i.c.) route of infection in DBA/2 mice. Whereas the highly pathogenic strain HSV-1 WAL, used as a control, led to lethal encephalitis by any mode of infection at 1 × 103 p.f.u./mouse, HSV-1 A N G proved to be non-lethal and did not cause signs of illness when applied intraperitoneally, intravenously, intravaginally, orally, or via the foot pads. However, i.c. infection with HSV-I A N G led to lethal encephalitis with very high efficiency. The LDs0 for i.c. inoculation of HSV-1 A N G was close to 1 p.f.u. (Fig. 1). With some individual HSV-1 A N G virus stocks, it appeared that even less than 1 p.f.u. (titre determined in cell culture) might result in lethal infection after i.c. injection. This effect could be explained by some HSV-I particles non-infectious in cell culture being able to replicate in mouse brain. The physical to infectious particle ratios of individual HSV-1 pools used in this study normally ranged between 10 : 1 and more than 100 : 1. (Schr6der & Urbaczka, 1978). Fig. 1 shows that the time of death clearly depended on the virus dose. As proof of the viral aetiology of the lethal outcome of i.c. inoculation of very low doses, virus titres were determined in the central nervous system (CNS). Titres in the brains of mice that eventually died after i.c. infection ranged between 5 × 103 and 1 × 104 p.f.u, per organ. In the spinal cord, up to 105 p.f.u, per animal were found. Infectious virus was not demonstrable in the CNS of survivors. Histological examination of spinal cord, spleen, liver, kidney, thymus, lung, heart, intestine and testicles from DBA/2 mice after i.c. infection with HSV-1 A N G failed to show major alterations in these organs. Focal haemorrhages around the corpus callosum, single cell necrosis of the pyramidal cells, glia satellitosis and microfocal bleedings in the meninges with inflammatory infiltrations suggested that the animals died from encephalitis or meningoencephalitis. All signs of myelitis were absent from tissue of the brain, the spinal cord and the dorsal root ganglia. In control animals injected i.e. with saline, haemorrhages in the brain indicated the site of injection, but other histological signs of the virus infection were absent. Primary replication of HSV-1 ANG in the recipient mouse and the spread of infection by the lethal and the non-lethal strains The usual pattern of pathogenesis by HSV-1 A N G could be explained by assuming that this virus strain is not able to replicate at the site of primary infection and therefore would not reach the cells of the CNS. In tissue culture, we demonstrated that HSV-1 A N G could be propagated in mouse primary fibroblasts and that it was able to replicate at the body temperature of the mouse. To compare the spread of infection of HSV-1 A N G to that of HSV-1 WAL, virus titres were determined in the organs of i.p.-injected DBA/2 mice. Fig. 2 shows the kinetics of virus replication in the peritoneal exudate and the spleen. These two organs turned out to be the main Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 08:27:14 318 G. K/JMEL I I I I 1 2 3 4 I AND (b) OTHERS I I I I I I i 3 4 5 6 7 e-~ _o 5 0 2 Time post-infection (days) Fig. 2. Spread'of infection with (a) HSV-1 ANG and (b) HSV-1 WAL in DBA/2 mice. Groups of four to six mice were infected intraperitoneallywith 4 × 105 p.f.u. At the times indicated, mice were dissected and virus titres determined by independent titration of samples. The bars indicate the standard error of the virus content per organ of peritoneal exudate (V), spleen (O), brain ([]) and spinal cord (11). reservoir of virus in the mouse. In other organs (thymus. liver, kidney, lung, heart and lymph nodes), virus was absent or ranged between 5 and 40 p.f.u, per organ in the first 6 days after infection for both virus strains. It was obvious that i.p. infection of DBA/2 mice with HSV-1 A N G is restricted, because after the fifth day post-infection, virus could not be detected in any of the mouse organs. The i.p. infection with HSV-1 W A L led to considerably higher titres (see Fig. 2b) in the peritoneum and the spleen but not in the other organs tested. The course of infection paralleled that of the nonlethal strain as the virus was eliminated by the fifth day from peritoneum and spleen. Increasing amounts of HSV-1 W A L were, however, found in C N S tissue 4 to 7 days after infection. The difference in pathogenicity between the two virus strains is therefore not due to a general restriction of primary replication. W e tested whether a major difference in systemic transport of the virus would correlate with the pathogenicity differences. After infection with either HSV-I strain, virus could not be demonstrated by direct titration of blood or serum 1, 2, 3, 4 or 5 days after infection. Thus, we are faced with the somewhat contradictory p h e n o m e n a that a HSV-1 strain replicates to high titres in mouse organs after systemic or peripheral infection without consequent lethal encephalitis, but that a few infectious virus particles invariably lead to lethal encephalitis if administered intracerebrally. In the following parts of this section, studies of the possible role of host defence mechanisms involved in the block o f the spread of HSV-1 A N G infections to the C N S are described. Infection of immunoincompetent mice with HSV-1 ANG To investigate the role of age-dependent defence mechanisms, i m m u n o i n c o m p e t e n t newborn mice were tested for susceptibility to HSV-1 A N G infection. For this study we used the C57BL/6 strain, because of its higher resistance to HSV (Zawatzky et al., 1982 a). C57BL/6 mice less than 7 days old were found to be susceptible to i.p. infection with HSV-1 A N G . Although the exact value of the LDs0 was not established, less than 103 p.f.u, were found to be sufficient to kill all of the animals (Table 1). The mechanism of the process limiting the spread of HSV-1 A N G into the C N S after systemic or peripheral infection therefore depends on elements of the defence system which are absent in newborn mice. Homozygous nude mice (nu/nu) and, as control, heterozygous mice (nu/+) (in each case I0 animals per group) were tested for their susceptibility to i.p. infection with 4 x 106 p.f.u. HSV-1 A N G . Only about 1 0 ~ of the mice died in either group between 2 and 21 days after infection. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 08:27:14 HS V strains differing in pathogenicity for mice 6 I I 1 2 I I I I 1 3 4 5 6 7 319 5 4 ,-" 4 3 1 Time post-infection (days) Fig. 3. Spread of HSV-l ANG after immunosuppression with cyclophosphamide of DBA/2 mice. Following injection of 150 mg/kg cyclophosphamide mice were injected with 4 x 10s p.f.u. intraperitoneally. At the time points indicated, mice were dissected and virus titres determined by independent titration of samples. Mean and standard error of virus titres per organ are given in the figure for four to six mice per time point; parallel blood samples were pooled. ~', Peritoneal exudate; O, spleen; O, liver; A, blood; m, brain. Table 1. Mortality of newborn and adult C57BL/6 mice following i.p. infection with HSV-1 ANG Expt. no. Age (days) 1 56 2 6 6 6 Dose used for infection (p.f.u./animal) Deaths/total number per group 2 x 106 0/10 2 × 104 6/6 2 x 103 6/6 2 x 102 2/6 Titration of the organs of those homozygous mice which died between these days failed to show infectious virus in the brain or spinal cord. These results indicate that the resistance against HSV-1 A N G is not dependent on mature T-lymphocytes. Infection of adult mice with HSV-1 ANG following immunosuppression F o r experimental immunosuppression D B A / 2 mice were treated with 150 mg/kg cyclophosphamide (Rager-Zisman & Allison, 1976). All of the immunosuppressed but uninfected animals survived whereas after i.p. infection with 1 x 105 p.f.u. HSV-1 A N G , all of the animals died. To examine the influence of immunosuppression on the course of infection, some of the infected animals were killed at appropriate time points, dissected and examined for the presence o f infectious virus in brain, spinal cord, liver, spleen and in the blood. Figure 3 shows that cyclophosphamide exerted a prominent effect on the kinetics of the infection. The drug induced a considerable increase in virus replication, particularly in the liver, peritoneal cavity and spleen. Titration of blood samples showed a distinct viraemia. However, little infectious virus was recovered from C N S tissue. The restriction of infection with HSV-1 A N G at the site of p r i m a r y infection and the spleen can clearly be overcome by immunosuppression. Unfortunately, cyclophosphamide changes the pathogenesis so much that no conclusion can be drawn about differences between the two virus strains, nor about a correlation between immunosuppression and the block in spreading o f HSV1 A N G to the CNS. Injection of 2.5 g/kg silica was used to suppress macrophage activity in D B A / 2 mice. The pretreated animals were infected i.p. 24 h later with 1 × 105 p.f.u. HSV-1 A N G . In this Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 08:27:14 320 G. KfJMEL AND OTHERS Table 2. Natural killer cell activity* of C57BL/6 mice after i.p. injection of H S V - I W A L or HSV-1 A N G Virus Effector cell origin ~ r strain Spleen Peritoneal cavity WAL ANG Uninfected 44 42 20 33 35 3 * Expressed as ~ specific lysis. Table 3. In vitro sensitivity of HSV-1 strains A N G and W A L to interferon in the L-929 cell line, or in mouse embryo fibroblasts (MEF) derived from DBA-2 or C57BL/6 mice* L-929 F Virus HSV-I A N G HSV-1 W A L - - - A _ _ MEF-DBA , . IFN r A - MEF-C57 ~ ( . ~ . K IFN - 2-0 × 106 2.5 x 104 3.3 × l 0 s 4.5 x 104 4.0 x 105 3"1 × 100 6.5 x 104 4.5 x 105 7.5 × 104 4.0 × 105 • IFN 2.0 x 104 2-8 x 104 * The values s h o w n are the progeny virus yield, g i v e n as the m e a n from three, 60 m m , Petri d i s h e s infected in parallel at an m.o.i, of 2. T h e r e were 2-5 x 106 L-929 cells or 1-6 × 106 M E F cells p e r dish. C u l t u r e s were p r e t r e a t e d w i t h 500 I U interferon ( I F N ) per m l m e d i u m . experiment, only 2 out of 15 animals died. Thus, we conclude that the mechanism limiting the spread of HSV-1 ANG into the CNS is not dependent on macrophage activity. Analysis o f N K cell activity after injection o f H S V - 1 A N G Major differences in the pathogenicity patterns of HSV-1 strains A N G and WAL could possibly be due to a difference in the capacity to induce N K cells. The NK cell activity induced after i.p. infection with HSV-1 A N G and the lethal strain HSV-1 WAL was assayed in C57BL/6 mice 24 h after i.p. infection with 1 x l0 s p.f.u, Table 2 shows that there were no differences in NK cell activity regardless of whether spleen ceils or peritoneal exudate cells (PEC) were examined. Induction of and sensitivity to, interferon The observed difference in the pathogenicities of HSV-1 A N G and HSV-I WAL could be a consequence of either a difference in interferon induction or different sensitivities of the virus strains to interferon. Primary embryonic mouse fibroblasts were cultured from the strains DBA/2 and C57BL/6. Table 3 shows that strains A N G and WAL replicated with the same efficiency in either of the two primary cell cultures or in the mouse cell line L-929 used as a control. Pre-treatment of either cell culture with an interferon dose of 500 IU/ml culture medium resulted in a reduced yield of progeny virus in both A N G and WAL. In each of the cell systems used, the sensitivity of the two virus strains to interferon proved to be essentially the same. In a mixed in vivo-in vitro approach, virus was injected i.p. and after one day PEC were recovered and cultivated without further addition of virus for 24 h. Subsequently, interferon titres in the tissue culture supernatant were determined. No difference between the virus strains was observed (Table 4). DISCUSSION Resistance of mice to peripheral infection with HSV is controlled by host gene functions (Lopez, 1975). Thus, it was important that our previous observation that HSV-1 A N G is nonpathogenic for C57BL/6 and DBA/2 mice (Schr6der et al., 1981) be extended to additional inbred mouse strains, including some that are known to be exquisitely sensitive to i.p. infection by other HSV strains (Kirchner et al., 1978). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 08:27:14 H S V strains differing in pathogenicity for mice 321 Table 4. Interferon production in peritoneal exudate cell cultures of C57BL]6 mice after i.p. infection of pathogenic HSV-1 W A L and non-pathogenic HSV-1 A N G Infecting virus HSV-I WAL HSV-I ANG Mock-infected Interferon titre (IU/mlmedium) 450 420 10 One might expect that resistance of mice to infection with HSV-1 A N G is expressed at the level of the target cells of lethal virus infection, i.e. the brain cells. We have shown, however, that HSV-1 A N G causes a lethal encephalitis after i.c. infection. Furthermore, following i.p. infection, a considerable amount of HSV-1 A N G can be found in the peritoneal cavity. Thus, HSV-1 A N G obviously is not non-pathogenic in a strict sense. Virus titres of strain A N G in the peritoneum and in the spleen are of a similar order of magnitude to those observed for HSV-1 WAL, indicating that the initial replication of HSV-1 A N G in vivo is not restricted. However, its transport to the brain or its propagation in the central nervous tissue seem to be efficiently blocked by a host defence mechanism. Previous work in our laboratory on infection of mice with HSV-1 WAL has suggested that interferon plays a critical role in the reistance of C57BL]6 mice to HSV (Zawatzky et al., 1981). This conclusion is based on the correlation observed between high titres of early interferon at the local infection site and high virus resistance (Zawatzky et al., 1982 b). DBA]2 mice did not show this type of early interferon production. In the present study, however, we found that DBA/2 mice nevertheless resist even high doses of HSV-1 ANG. Series of in vitro and in vivo experiments showed that HSV-1 A N G does not induce a more efficient interferon response than HSV-1 WAL in DBA mice or that the non-lethal virus has a higher sensitivity to interferon. A variety of additional approaches was utilized to study the role of defence mechanisms in the lack of pathogenicity of HSV-1 ANG. First, it appeared that macrophages were not playing a major role, since treatment of mice with high doses of silica did not remove resistance to HSV-1 ANG. Immunosuppression with cyclophosphamide led to a dramatic enhancement of the invasiveness of HSV-1 A N G and to the death of the animals. It seems therefore safe to assume a major role of immune control in the restriction of primary replication of HSV-1 at the site of the inoculation and in the spleen. It cannot be decided from the data whether the second step of the course of infection, transport to and propagation in CNS tissue, is under the control of the immune system. Newborn mice up to 10 days of age are also fully susceptible to infection with HSV-1 ANG. Thus, the relevant defence mechanism that restricts the infection with HSV-1 A N G appears to be absent during the first 10 days of life. Since we have previously shown that N K cell activation by HSV is defective in newborn mice (Zawatzky et al., 1982a), we have measured activation by HSV-1 A N G to test the assumption that HSV-I A N G is a better N K cell inducer than HSV-I WAL. This was found not to be the case, however, so we assume that N K cells do not play a major role in the restriction of HSV-I ANG. We have demonstrated that nu]nu mice have a pronounced resistance to HSV-1 ANG. Therefore we conclude tentatively that mature T-cells do not play a role in the resistance of mice to infections with this strain of HSV. Nude mice are not only deficient in cell-mediated immunity but also lack the capacity to produce certain antiviral antibodies (Burns et al., 1975). Thus, nude mice are unable to produce IgG antibodies against HSV (Hilfenhaus et al., 1981). Our data with HSV-1 A N G in nude mice therefore suggest that the IgG response of the host is not responsible for the observed inhibition of pathogenicity. In this context, it has to be remembered that primary virus replication is of the same order of magnitude in the peritoneal cavity and in the spleen and that no viraemic process was found for HSV-1 A N G or for the pathogenic strain HSV-I WAL. Thus it appears that the blocking mechanism which prevents HSV-1 A N G from reaching the brain is not an early but, rather, a relatively late defence mechanism. The studies of Kastrukoff et al. (1981) have indicated that Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 08:27:14 322 G. KUMEL AND OTHERS a n t i b o d i e s o f the I g M class are p r o d u c e d w i t h i n a few days o f i n f e c t i o n o f m i c e by H S V . Since the p r o d u c t i o n o f I g M antibodies in n u d e m i c e is n o r m a l and does not require h e l p e r T-cells, one possible e x p l a n a t i o n i n v o l v i n g the i m m u n e system would be t h a t HSV-1 A N G , in c o n t r a s t to p a t h o g e n i c HSV-1 isolates, is a m o r e effective i n d u c e r o f I g M antibodies. O n the o t h e r hand, unspecific, i.e. n o n - i m m u n e , processes h a v e to be c o n s i d e r e d as a possible cause for the lack of p a t h o g e n i c i t y o f HSV-1 A N G . Differential r e p l i c a t i o n in special t a r g e t cells, differential uptake by p e r i p h e r a l neurons or an unspecific, a g e - d e p e n d e n t process are e x a m p l e s o f such n o n - i m m u n e m e c h a n i s m s . But diffferential r e p l i c a t i o n in m a c r o p h a g e s (J. Briicher & H. K i r c h n e r , unpublished), a difference in v i r a e m i c transport, i n t e r f e r o n - s e n s i t i v i t y or its induction, or N K cells all seem to be excluded. Recently, an isogenic v a r i a n t o f HSV-1 A N G has been isolated in this l a b o r a t o r y w h i c h p r o v e d to be highly p a t h o g e n i c for m i c e after p e r i p h e r a l i n f e c t i o n ( K a e r n e r et al., 1981). It displays distinct alterations in its g e n o m e w h e n c o m p a r e d to the original HSV-1 A N G . A c o m p a r a t i v e analysis of HSV-1 A N G and its p a t h o g e n i c v a r i a n t , w i t h r e g a r d to virus s p r e a d and their influence on various host d e f e n c e m e c h a n i s m s , is in progress. REFERENCES ANDERVONT,H. B. (1927). Activity of herpetic virus in mice. American Journal of Hygiene 14, 383-393. BECK, J., ENGLER, H., BRUNNER, H. & KIRCHNER, H. (1980). Interferon production in cocultures between mouse spleen ceils and tumor cells. 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