Vol. 21, No.3
INFECTION AND IMMUNITY, Sept. 1978, p. 764-770 0019-9567/78/0021-0764$02.00/0 Copyright C 1978 American Society for Microbiology
Printed in U.S.A.
Susceptibility of Mice to Acute and Persistent Measles Infection P. A. NEIGHBOUR, B. RAGER-ZISMAN,t AND B. R. BLOOM* Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461 Received for publication 2 May 1978
Intracerebral inoculation of neonatal mice with the Edmonston strain of measles virus produced an acute, lethal encephalitis and thymic dysplasia in susceptible mice. There was an age-related development of resistance to infection. This resistance was strain-dependent and appeared to be associated with the extent of virus growth in the brain. Studies on the genetic basis for susceptibility, using hybrid and backcross mice, revealed that the principal determinant of host resistance to acute infection was a dominant gene or genes which segregated independently of the H-2 complex. A small number of survivors of the acute infection showed persistence of measles virus antigens in the cerebellum and spleen for up to 2 months after inoculation. However, the low frequency of this persistence indicated that, at this time, intracerebral inoculation of neonatal mice with the Edmonston strain of measles virus constitutes a difficult model for the study of persistent measles infection. Several slow neurological diseases of humans are currently believed to result from the persistence of viruses (6). Perhaps the most direct evidence of such a causal relationship is the association between subacute sclerosing panencephalitis (SSPE) and measles virus. Patients with this disease exhibit elevated levels of antimeasles antibodies in the serum and cerebrospinal fluid (5), and a measles-like virus has been isolated from their brain tissues (11) and lymph nodes (12). Although this virus can be neutralized by anti-measles serum, it has been found to have a genome RNA which is 10% larger than that reported for measles virus (9), and an M protein with an altered electrophoretic mobility
(29).
The association between measles and multiple sclerosis (MS) is more tenuous. The disease probably has a multiple etiology (2, 14), and at present there has been no compelling evidence that an infectious agent might cause this disease. Epidemiological investigations favor this etiology, and MS has been causally linked with Sendai, herpes-, rabies, and measles viruses. Of these, measles virus has received perhaps the most attention following the original observation of Adams and Imagawa (1) of elevated measles antibody levels in the sera and cerebrospinal fluid of MS patients. Defects in the cell-mediated immune responses to measles virus have been reported for individuals suffering from MS t Present address: Ben Gurion University of the Negev, Beer-Sheva, Israel.
(26, 27). Recently, measles was reported to be observed by immunofluorescence and by cocultivation in the jejunum of chronic MS patients (21, 22), although this observation has yet to be confirmed. Factors other than an infectious agent appear to be involved in the etiology of MS. Of particular interest is the finding of a marked genetic predisposition to MS, with a high correlation between the incidence of the disease and certain histocompatibility (HLA) types (19). For example, in a study in Western Europe, 70% of MS patients were found to express the HLA specificity, DW2, compared with only 16% of normal individuals (13). One of the problems in understanding the pathogenesis of SSPE and MS has been the lack of suitable animal models. Measles virus is a human pathogen, and it has proven difficult to develop appropriate models for acute and chronic measles infection in laboratory animals. Apart from experiments in nonhuman primates with SSPE (30), most studies have involved inoculation of weanling or neonatal hamsters with wild-type measles (28), SSPE virus (3), or temperature-sensitive mutants of measles (10). The severity of disease observed in these studies was age dependent. Newborn hamsters developed an acute, fatal, giant cell encephalitis, whereas a chronic inclusion cell encephalitis, similar to human SSPE, occurred in weanling animals. The existence of a multitude of inbred strains of mice would enable investigation of any genetic restriction of susceptibility or resistance 764
VOL. 21, 1978
SUSCEPTIBILITY OF MICE TO MEASLES INFECTION
to infection with measles. Griffin et al. (7) observed an age-dependent susceptibility to an acute, lethal infection in neonatal BALB/c mice, but did not investigate the influence of host genotype on the outcome of the disease. The present report represents a preliminary study in mice of the genetic basis for susceptibility to the acute, lethal encephalitis which follows intracerebral (i.c.) inoculation of measles virus. In addition, it will be indicated that under appropriate conditions, persistent infection beyond the period of acute infection can be achieved at a low frequency in surviving animals. MATERIALS AND METHODS Mice. Inbred mice of the following strains were used to provide inbred, F, hybrid, and backcross neonates: C3H/HeJ, C3HeB/FeJ, C3H/HeSn, AKR, BALB/c, DBA/2, C57BL/6, C57BL/10 (BO), SJL/J, A/J (all obtained from the Jackson Laboratory, Bar Harbor, Me.) and CBA/Ca (from the Animal Division of the Clinical Research Centre, London, England). In addition, mice of various congenic strains (all obtained from F. Lilly) of BALB/c (BALB.B and BALB.K), of C3H (C3H.Sw), and of C57BL/10 (B1O.Br) were also used in this study. All matings were carried out in this laboratory to provide neonates of the required ages. Virus. The Edmonston strain of measles virus (obtained from B. Fields) was serially passaged in vivo through the brains of neonatal hamsters. After double plaque purification in CV-1 cells, the virus was passed once through neonatal mouse brain, and then virus stocks were prepared by two passages at a low multi-
plicity of infection of 0.01 in CV-1 cells. The virus was harvested by alternately freezing and thawing the infected cell monolyer and medium at 72 h after inoculation, followed by centrifugation at 1,000 x g for 10 min at 40C to remove cell debris. The virus stock, which had a titer of 1.5 x 106 plaque-forming units (PFU) per ml, was stored frozen at -700C until required. Virus titration. Virus samples were titrated by using the semi-microplaque method (23) with CV-1 or Vero cells as indicators. Inoculation of mice. For all experiments, unless otherwise stated in the text, each mouse received an i.c. inoculation of 0.03 ml of undiluted virus stock containing 4.5 X 104 PFU of measles virus. Histology. Tissues were fixed in formal-saline for 48 h, embedded in paraffin wax and microtome sectioned. Sections were stained with hematoxylin and eosin.
Immunofluorescence. Freshly excised tissues were "snap-frozen" in cold (-750C) iso-pentane, cryostat sectioned at 4 to 6 um, and either air-dried or fixed in cold (40C) acetone. Sections were stained with a fluorescein isothiocyanate-conjugated hyperimmune rabbit anti-measles virus serum (hemagglutination inhibition titer, 1/1,280), and rhodamine-labeled bovine serum albumin was used as counterstain. Stained sections were viewed on a Leitz Orthoplan fluorescence microscope with epi-illumination at a wavelength of 490 .m.
Detection of infectious virus in tissues. Brain,
765
thymus, liver, spleen, kidneys, and peripheral lymph nodes were removed from acutely infected mice at intervals after inoculation, homogenized with a ground glass homogenizer in 1 ml of Eagle minimum essential medium containing antibiotics and 5% (vol/vol) fetal calf serum, and titrated as described above. To detect the persistence of measles virus, survivors of the acute infection were killed at intervals, and the same organs were removed. Cell suspensions were prepared by mechanical disruption in Eagle minimum essential medium and these were titrated directly; cocultivated with monolayers of Vero indicator cells; or fused with Vero cells in suspension using polyethylene glycol (8), seeded in culture flasks, and cultured until confluent. Cocultivation and fusion cultures were passaged at least twice before discard and were examined daily for measles-induced cytopathic effect. H-2 haplotype determination. The haplotype of backcross progeny mice was determined by a microhemagglutination assay using washed mouse erythrocytes and a specific anti-H-2' immune serum (from D. C. Shreffler).
RESULTS Acute infection. i.c. inoculation of 1- to 2day-old neonatal C3H/HeJ, CBA, and BALB/c mice with measles virus produced an acute lethal disease in all mice. The clinical signs of infection were first apparent 5 to 7 days after inoculation when the mice became hyperactive for about 24 to 48 h. This was followed by a period of ataxia, paralysis, and finally by death. Infected mice exhibited some growth retardation when compared with uninfected, age-matched control mice. Histopathological lesions were observed in the cerebral cortex and hippocampus. Focal lesions of inflammation and necrosis with infiltrating mononuclear cells were frequently surrounded by giant syncytial cells, comprised primarily of neurons. Some focal necrosis of neurons with swelling of nuclei and vacuolation was also observed. The first foci were detected 4 days after inoculation, and the number of lesions increased progressively. After a further 3 to 5 days, the histopathology was severe, and infectious foci were spread throughout the cerebral cortex and hippocampus. Immunofluorescent staining showed that in acutely infected mice, measles viral antigen appeared initially in the cerebral cortex 4 days after inoculation. Infected cells occurred in discrete foci, and the antigen was detected both on the cell membrane and as cytoplasmic inclusions. Nuclear inclusions of viral antigen were also observed in some of these cells. Staining was intense throughout the cerebral cortex 9 days after inoculation, and foci appeared in the hippocampus. Maximum titers of virus were recovered from the brains of infected CBA mice 8 to 10 days after inoculation, which is consistent with the appearance of viral antigen and histopathology (Fig. 1). Similar re-
766
INFECT. IMMUN.
NEIGHBOUR, RAGER-ZISMAN, AND BLOOM
Age- and strain-related susceptibility of inbred mice to acute infection. Mice from eight inbred strains were all found to be highly susceptible to acute infection when inoculated with a high dose of measles (4.5 x 104 PFU) ,D 6D within 2 days of birth (Table 1). However, a variation in susceptibility to virus dose was apparent in some of these strains during the first 2 days after birth when the titer of virus required 'LU 5.0 to produce lethal infection was determined for each strain (Table 2). When mice from some of these strains were inoculated with measles at different ages, there was an age-dependent var4.0iation between strains in susceptibility to infection (Table 3). Although C3H/HeJ mice re2 4 6 8 10 12 14 mained highly susceptible to lethal infection at DAYS AFTER INOCULATION 3 to 4 days of age, BALB/c and C57BL/6 exFIG. 1. Virus titers in brain homogenates at inter- hibited an intermediate susceptibility, and SJL vals after i.c. inoculation of 1- to 2-day-old CBA mice mice were totally resistant. CBA mice and 0
with measles virus. Each point represents the mean titer (± standard error of the mean) from a minimum of three mice on each of the days indicated.
TABLE 1. Mortality rates (%o) in 1- to 2-day-old mice of various inbred strains after i.c. inoculation with measles virus
sults were obtained with C3H/HeJ mice. No infectious virus or viral antigen was detected in the thymus, liver, spleen, kidney, or peripheral lymph nodes of any of the infected mice. To determine the lethal dose of virus for 50% of inoculated mice (LDrA), 1- to 2-day-old CBA mice (14 to 21 mice for each dose) were inoculated i.c. with serial 10-fold dilutions of the stock virus and observed daily for death. Mice which died within 3 days of inoculation were excluded from the data, since death was assumed to have resulted from inoculation trauma. The LD5o was calculated from the mortality rates by the method of Reed and Muench (24), and was found to be 27 PFU per mouse. Inoculation of 1- to 2-day-old mice with measles virus by the intraperitoneal or intranasal routes did not produce any detectable disease or infection.
Strain
No. of mice H-2haplotype inoculated
k k k d d b
C3H/HeJ CBA AKR BALB/c DBA/2 C57BL/6 SJL A/J
s a
15 28 5 10 8 12 8 5
Mortality rate (%)
100 100 100 100 100 91.7 100 100
TABLE 2. LDro of measles virus for 1- to 2-day-old mice of different strains Strain LDr, 10,000
TABLE 3. Mortality rates (%) in neonatal mice of various inbred strains after i.c. inoculation with measles virus at different ages Strain
C3H/HeJ BALB/c
H-2type haplo-
k
1-2a
100
(15/15)b d
100
3-4 100
(19/19) 40.0 (6/15)
5-6
7-8
9-10
11-12
18-20
92.3
85.7
44.4
22.7
0
(48/52)
(12/14)
(4/9)
(5/22)
0
(0/14)
NDc
ND
ND
(0/9) 0 (0/18) ND
0 (0/7 ND
ND
0 (0/6) ND
14.8 (4/27) b 91.7 57.1 0 C57BL/6 (11/12) (8/14) (0/15) s SJL 100 0 0 (8/8) (0/13) (0/15) a Age (days) at inoculation (4.5 x 104 PFU per mouse). ' No. dead/no. inoculated. 'ND, Not done.
(10/10)
Mortality rate (%)
ND
SUSCEPTIBILITY OF MICE TO MEASLES INFECTION
VOL. 21, 1978
other C3H sublines, namely C'3HeB/FeJ and C3H/HeSn, were also found to be highly susceptible (data not shown). In additi( )n, the duration of the acute disease and the day of death varied according to their relative susc eptibilities (Fig. 2). To determine whether the va rying susceptibilities exhibited by these strains were correlated with the extent of viral replicatii on, 5- to 6-day100
C3H
80 60 40 20
_;_S.
'
______t~~~~ 0
LuS
100- C57BI/6 80
60 I.-
40 20 (
100
-
-L
-
-
,
'1I-
SJL
80
60
40 20 0
7
14
21
.1___L.......J 35
28
42
DAYS AFTER INOCLJLATION
FIG. 2. Cumulative mortality raltes in four inbred strains after i.c. inoculation hwith measles virus at I to 2 days (solid line) and 3 to 4 days (dotted line) of age. mouse
767
old C3H, BALB/c, and SJL mice were inoculated with the standard inoculum of measles virus, and their brains were examined for evidence of productive infection at weekly intervals. Table 4 shows that considerable viral replication had occurred in the brains of C3H mice by 7 days after inoculation, there being high titers of infectious virus in most of the brains examined and numerous foci of viral antigen throughout the cerebral cortex. After a further 7 days, no virus was recovered from surviving mice, although a few scattered fluorescent cells were seen in the cerebral cortex. The brains from more resistant BALB/c mice exhibited less evidence for viral infection. Virus was recovered from only one brain, and only a few foci of viral antigen were observed in the cerebral cortex of some mice. By 14 days after inoculation, viral infection appeared as only a few positively stained cells in the Purkinje layer of the cere-
bellum. None of the highly resistant SJL mice showed infection of their brains at any time after inoculation. No virus was recovered, or immunofluorescence observed, in the brains of mice from any of these strains at 21 or 28 days after inoculation. Growth retardation during acute infection. Inoculation of 5- to 6-day-old C3H mice with a lethal dose of measles virus produced a marked retardation in growth before death. This apparent "runting" was found to be associated with thymic dysplasia (Fig. 3). Histological examination of the thymuses from these mice re-
vealed an absence of clear cortical and medullary junctions, and many pyknotic nuclei and karyorrhectic cells were observed. Viral antigen was not detected in these organs by immunofluores-
and infectious virus could not be covered.
cence,
re-
TABLE 4. Production of infectious virus or viral antigen in the brains of C3H, BALBIc, and SJL mice after i.c. inoculation with measles virus Virus antigen
Infectious virus Strain'
after inDays oculation
FrequencyCb
Mean titer (logoPFU/g)
4.3 7 6/7 0 0/4 14 0 0/4 21 0 28 0/2 4.2 7 1/4 BALB/c 0 0/4 14 0 21 0/6 0 28 0/4 0 7 0/7 SJL 0 14 0/7 0 21 0/5 0 0/4 28 a Mice (5 to 6 days old) were inoculated i.c. with 4.5 X ' No. of positive brains/no. examined.
C3H/HeJ
FFrequency'
Location
Numerous foci in cerebral cortex 6/7 Few discrete cells in cerebral cortex 2/4 Negative 0/4 Negative 0/2 Several foci in cerebral cortex 2/4 Few cells in Purkinje layer 4/4 Negative 0/6 Negative 0/4 Negative 0/7 Negative 0/7 Negative 0/5 Negative 0/4 104 PFU of measles virus.
768
INFECTr. IMMUN .
NEIGHBOUR, RAGER-ZISMAN, AND BLOOM
Genetic basis of resistance. Since mice with the H-2k haplotype (C3H) were susceptible to lethal measles infection, and those with the H-2' haplotype (SJL) were resistant, experiments were performed to determine whether the major histocompatibility gene complex controlled host susceptibility to this virus. F1 (C3H x SJL) hybrid mice, 5 to 6 days old, which were heterozygous for the s and k alleles were found to be totally resistant (Table 5). Progeny derived from a backcross of F. (C3H x SJL) hybrids with the susceptible C3H parent exhibited an intermediate susceptibility (56%) when inoculated at 5 to 6 days of age. Surviving mice were typed for their H-2 haplotype, and there was found to be an equal distribution of mice with H-2 /k and H-2k1k (Table 5). Therefore, resistance to acute measles infection appears to segregate independently of the H-2 complex in these two strains. To further investigate the role played by the major histocompatibility gene complex in controlling susceptibility to lethal measles infection, 5- to 6-day-old mice of various congenic strains
were inoculated with this virus. BALB.B (H-2b from C57BL/10) and BALB.K (H-2' from C3Hf/An) did not differ in their susceptibilities from BALB/c (H-2d) (Table 6). However, C3H.Sw (H-2b) mice were significantly more resistant (P < 0.001, chi-square test) than congenic C3H (H-2k) mice, and B10.Br (H-2k) were more susceptible than congenic B10 (H-2b) mice (Table 6). Thus, the inoculation of H-2 congenic mice suggested that in some strains the H-2 complex might modify expression of the gene or genes which determine resistance. Persistent infection. In Table 4 it was indicated that viral antigen could be detected in the brains of acutely infected C3H and BALB/c mice up to 14 days after inoculation. To determine whether neonatal i.c. inoculation with measles virus could lead to a persistent infection, mice which had survived the acute episode were killed at intervals, and their organs were examined for viral antigen by immunofluorescence or infectious virus by cocultivation or cell fusion techniques. Of 18 C3H, 26 BALB/c, 12 C57BL/6, and 18 SJL mice killed between 1 and 14 months after inoculation, viral antigen was found in only 70 3 BALB/c mice, at 58 days after inoculation. In these mice, a few discrete fluorescent cells were 601observed in the cerebral cortex and along the Purkinje layers of the cerebellum. In addition, a 501few fluorescent foci were observed in the spleens of two of the three mice. Fluorescent staining was blocked with unconjugated rabbit anti-meaE 40 sles serum, but not by nonimmune rabbit serum, confirming the specificity of this staining for 030 measles antigen. No infectious virus could be recovered from cells obtained from brain, thy20u-I mus, liver, or spleen of any of the survivors of the acute infection by cocultivation or by poly10 ethylene glycol fusion with permissive Vero cells. 21 3 5 7 10 12 14 17 DISCUSSION DAYS AFTER INOCULATION The results of these experiments show that FIG. 3. Thymic dysplasia after i.c. inoculation of 5- to 6-day-old C3H mice with measles virus: unin- the Edmonston strain of measles virus is neurofected mice (0); infected mice (0). virulent for newborn mice. i.c. inoculation during uJ
-
TABLE 5. Relationship between H-2 and susceptibility to lethal measles infection No. of
Strain" Strain'
iicN in miocelam-
H-2 haplotype frequency' of inMortality rate
C3H SJL
(%
oculated mice
s/s
52 92.3 0 15 0.0 100 18 0.0 0 F,(C3H x SJL) 0 C3H x F. (C3H x SJL) 74 56.8 aMice (5 to 6 days old) were inoculated i.c. with 4.5 x bAll frequencies are theoretical unless indicated. Frequency determined by sera typing. d
Frequency calculated by subtraction.
s/k
k/k
H-2 haplotype frequency of surviving mice
s/s
s/k
k/k
0 100 0 0 0 100 100 0 0 0 51c 49d 104 PFU of measles virus.
0 0 100
100 0 0
54C
46d
VOL. 21, 1978
SUSCEPTIBILITY OF MICE TO MEASLES INFECTION
TABLE 6. Susceptibility of various congenic strains to lethal measles infection Strain'
H-2type haplo-
No. of mice inoculated
Mortality (%) rate
BALB/c BALB.B BALB.K C3H C3H.Sw B10
d 27 14.8 b 23 17.4 k 12 16.7 k 52 92.3 b 20 40.0 b 19 47.4 B10.Br k 70 70.0 a Mice (5 to 6 days old) were inoculated i.c. with 4.5 x 104 PFU of measles virus.
the first 48 h after birth produced an acute, lethal encephalitis in almost all mice. Many mice were runted, and this was probably a result of the thymic dysplasia which occurred in these mice. There was an age-related development of resistance to infection, and the rate of this development appeared to be strain dependent. The various inbred strains of mice used in this study could be designated as being either susceptible, resistant, or intermediate when inoculated after 2 days of age. However, even in the highly susceptible strains, the period of susceptibility was short-lived. The consequence of acute infection was clearly related to the extent of virus growth in the brain as shown by the recovery of infectious virus and by the appearance of viral antigen at intervals after inoculation. Susceptible C3H mice exhibited numerous foci of infection in the cerebral cortex, and virus was recovered from almost all of the mice inoculated. The extent of the infection was somewhat reduced in a strain of intermediate susceptibility, namely BALB/c, whereas totally resistant SJL mice failed to exhibit any viral growth in brain tissues. It is not known whether this reduction in virus growth results from increased immune responsiveness or whether it reflects different patterns of neurodifferentiation with loss of measles-permissive cells in resistant mice. Differences in susceptibility in various strains of inbred mice suggested that the major histocompatibility complex might be implicated in the control of susceptibility or resistance to acute measles infection. It is known, for example, that resistance to murine leukemia is controlled by a number of genetic loci, two of which, Rgv-1 (15) and Rfv-1 (4), are associated with H2. Rgv-1 maps proximally towards the K region of H-2 (15), and it has been suggested that this gene might be linked to the immune response (Ir) locus (18). Indeed, if the immune response is responsible for recovery from acute measles infection in resistant mice, an appropriate Ir-
769
associated gene might determine susceptibility or resistance to this virus. This is believed to be the case in mice for susceptibility to lymphocytic choriomeningitis virus disease (20). However, in a number of other systems, the major determinants of resistance are not associated with the major histocompatibility complex. For example, the two major determinants of susceptibility to Friend virus-induced leukemogenesis, called Fv-l and Fv-2, map on chromosomes 4 and 9, respectively (25). Resistance of mice to various myxoviruses is determined by a single dominant autosomal locus called Mx (16), and three nonH-2-linked genes are believed to control resistance of mice to herpes simplex virus infection (17). In the present study, the gene or genes determining resistance to measles were found to be dominant, since F. hybrids derived from susceptible (C3H) and resistant (SJL) parental strains were totally resistant to lethal infection. However, backcrossing of these resistant F, mice with the susceptible parental strain (C3H) showed that resistance segregated independently of H-2. Although the simplest interpretation of these data would suggest that a single, dominant, nonH-2-linked gene determines susceptibility or resistance, further testing of progeny from these F2 mice for their susceptibility to acute infection will be required to estimate the number of genes involved. Data obtained from the inoculation of certain congenic strains suggests that although the H-2 complex does not determine susceptibility or resistance, it might modify expression of the gene or genes involved. C3H.Sw mice, which were significantly more resistant than congenic C3H mice, have a resistant H-2 haplotype (H-28) on a susceptible C3H background. Conversely, B1O.Br were more susceptible than congenic B10 mice, and they have a susceptible H-2 haplotype (H-2k) on a resistant background. BALB/c congenic strains did not exhibit any significant changes in susceptibility despite their different H-2 haplotypes. Finally, because two slow neurological diseases of humans, SSPE and MS, have been associated with persistent measles infection, an attempt was made to determine whether Edmonston measles virus could persist in mice. A few survivors of acute infection did exhibit persistence of measles virus antigens in cells of the cerebellum and spleen, but the frequency was very low (3/74). We must conclude that, at the present time, i.c. inoculation of neonatal mice with this strain of measles virus constitutes an unsatisfactory model for the investigation of persistent measles infection. Perhaps with adaptation of this virus to murine nervous tissues,
770
NEIGHBOUR, RAGER-ZISMAN, AND BLOOM
immunosuppression, or passive transfer of measles antibody, the model could become more useful for the investigation of persistent measles infection. ACKNOWLEDGMENTS This study was supported by National Multiple Sclerosis Society grant 1006-B-2. We are grateful to Grace Ju for helpful discussions in preparation of this manuscript and to R. D. Barnes, Clinical Research Centre, London, England, for providing facilities for part of this investigation.
LITERATURE CITED 1. Adams, J. M., and D. T. Imagawa. 1962. Measles antibodies in multiple sclerosis. Proc. Soc. Exp. Biol. Med. 111:562-566. 2. Black, F. L 1975. The association between measles and multiple sclerosis. Prog. Med. Virol. 21:158-164. 3. Byington, D. P., and K. P. Johnson. 1973. Subacute sclerosing panencephalitis (SSPE) agent in hamsters. II. The neuropathology of acute and chronic infections. Exp. Mol. Pathol. 18:345-356. 4. Chesebro, B., K. Wehrly, and J. H. Stimpfiing. 1974. Host genetic control of recovery from Friend leukemia virus-induced splenomegaly: mapping of a gene within the major histocompatibility complex. J. Exp. Med. 140:1457-1467. 5. Connolly, J. H., I. V. Allen, L. J. Hurwitz, and J. H. D. Millar. 1967. Measles-virus antibody and antigen in subacute sclerosing panencephalitis. Lancet i:542-544. 6. Fucillo, D. A., J. E. Kurent, and J. L Sever. 1974. Slow virus diseases. Annu. Rev. Microbiol. 28:231-264. 7. Griffin, D. E., J. Mullinix, 0. Narayan, and R. T. Johnson. 1974. Age dependence of viral expression: comparative pathogenesis of two rodent-adapted strains of measles virus in mice. Infect. Immun. 9:690-695. 8. Hales, A. 1977. A procedure for the fusion of cells in suspension by means of polyethylene glycol. Somat. Cell Genet. 3:227-230. 9. Hall, W. W., and V. ter Meulen. 1976. RNA homology between subacute sclerosing panencephalitis and measles viruses. Nature (London) 264:474-477. 10. Haspel, M. V., R. Duff, and F. Rapp. 1975. Experimental measles encephalitis: a genetic analysis. Infect. Immun.12:785-790. 11. Horta-Barbosa, L, D. A. Fucillo, J. L Sever, and W. Zeman. 1969. Subacute sclerosing panencephalitis: isolation of measles virus from a brain biopsy. Nature (London) 221:974. 12. Horta-Barbosa, L, R. Hamilton, B. Wittig, D. A. Fucillo, J. L Sever, and M. L. Vernon. 1971. Subacute sclerosing panencephalitis: isolation of suppressed measles virus from lymph node biopsies. Science 173:840-841. 13. Jersild, C., T. Fog, G. S. Hansen, M. Thomsen, A. Svejgaard, and B. Dupont. 1973. Histocompatibility determinants in multiple sclerosis, with special refer-
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ence to clinical course. Lancet ii:1221-1224. 14. Johnson, R. T. 1975. The possible viral etiology of multiple sclerosis. Adv. Neurol. 13:1-46. 15. Lilly, F. 1970. The role of genetics in Gross virus leukemogenesis, p. 213-220. In R. M. Dutcher (ed.), Comparative leukemia research, 1969. Karger, Basel. 16. Lindenmann, J. 1964. Inheritance of resistance to influenza virus in mice. Proc. Soc. Exp. Biol. Med. 116:506-509. 17. Lopez, C. 1975. Genetics of natural resistance to herpesvirus infections in mice. Nature (London) 258:152-153. 18. McDevitt, H. O., M. B. A. Oldstone, and T. Pincus. 1974. Histocompatibility-linked genetic control of specific immune responses to viral infection. Transplant. Rev. 19:209-225. 19. McFarlin, D. E., and H. F. McFarland. 1976. Histocompatibility studies and multiple sclerosis. Arch. Neurol. 33:395-398. 20. Oldstone, M. B. A., F. J. Dixon, G. F. Mitchell, and H. 0. McDevitt. 1973. Histocompatibility-linked genetic control of disease susceptibility. J. Exp. Med. 137:1201-1212. 21. Pertschuk, L. P., A. W. Cook, and J. Gupta. 1976. Measles antigen in multiple sclerosis: identification in the jejunum by immunofluorescence. Life Sci. 19:1603-1608. 22. Prasad, I., J. D. Broome, L. P. Pertschuk, J. Gupta, and A. W. Cook. 1977. Recovery of paramyxovirus from the jejunum of patients with multiple sclerosis. Lancet i:1117-1123. 23. Rager-Zisman, B., and T. C. Merigan. 1973. A useful quantitative semimicromethod for viral plaque assay. Proc. Soc. Exp. Biol. Med. 142:1174-1179. 24. Reed, L. J., and H. Muench. 1938. A simple method of estimating fifty percent end points. Am. J. Hyg. 27:493-497. 25. Steeves, R., and F. Lilly. 1977. Interactions between host and viral genomes in mouse leukemia. Annu. Rev. Genet. 11:277-296. 26. Utermohlen, V., J. Farmer, J. Kornbluth, and M. Kornstein. 1977. The relationship between direct migration inhibition with measles antigen and E rosettes in normals and patients with multiple sclerosis. Clin. Immunol. Immunopathol. 9:63-66. 27. Utermohlen, V., and J. B. Zabriskie. 1973. A suppression of cellular immunity in patients with multiple sclerosis. J. Exp. Med. 138:1591-1596. 28. Wear, D. J., and F. Rapp. 1971. Latent measles virus infection of the hamster central nervous system. J. Immunol. 107:1593-1598. 29. Wechsler, S. L., and B. N. Fields. 1978. Differences between the intracellular polypeptides of measles and subacute sclerosing panencephalitis virus. Nature (London) 272:458-460. 30. Yamanouchi, K., N. Uchida, S. Katow, T. A. Sato, K. Kobune, F. Kobune, Y. Yoshikawa, H. Kodama, and A. Shishido. 1976. Growth of measles virus in nervous tissues. IV. Neurovimlence in wild measles and SSPE viruses in monkeys. Jpn. J. Med. Sci. Biol. 29:177-186.
INFECTION AND IMMUNITY, Sept. 1978, p. 764-770 0019-9567/78/0021-0764$02.00/0 Copyright C 1978 American Society for Microbiology
Printed in U.S.A.
Susceptibility of Mice to Acute and Persistent Measles Infection P. A. NEIGHBOUR, B. RAGER-ZISMAN,t AND B. R. BLOOM* Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461 Received for publication 2 May 1978
Intracerebral inoculation of neonatal mice with the Edmonston strain of measles virus produced an acute, lethal encephalitis and thymic dysplasia in susceptible mice. There was an age-related development of resistance to infection. This resistance was strain-dependent and appeared to be associated with the extent of virus growth in the brain. Studies on the genetic basis for susceptibility, using hybrid and backcross mice, revealed that the principal determinant of host resistance to acute infection was a dominant gene or genes which segregated independently of the H-2 complex. A small number of survivors of the acute infection showed persistence of measles virus antigens in the cerebellum and spleen for up to 2 months after inoculation. However, the low frequency of this persistence indicated that, at this time, intracerebral inoculation of neonatal mice with the Edmonston strain of measles virus constitutes a difficult model for the study of persistent measles infection. Several slow neurological diseases of humans are currently believed to result from the persistence of viruses (6). Perhaps the most direct evidence of such a causal relationship is the association between subacute sclerosing panencephalitis (SSPE) and measles virus. Patients with this disease exhibit elevated levels of antimeasles antibodies in the serum and cerebrospinal fluid (5), and a measles-like virus has been isolated from their brain tissues (11) and lymph nodes (12). Although this virus can be neutralized by anti-measles serum, it has been found to have a genome RNA which is 10% larger than that reported for measles virus (9), and an M protein with an altered electrophoretic mobility
(29).
The association between measles and multiple sclerosis (MS) is more tenuous. The disease probably has a multiple etiology (2, 14), and at present there has been no compelling evidence that an infectious agent might cause this disease. Epidemiological investigations favor this etiology, and MS has been causally linked with Sendai, herpes-, rabies, and measles viruses. Of these, measles virus has received perhaps the most attention following the original observation of Adams and Imagawa (1) of elevated measles antibody levels in the sera and cerebrospinal fluid of MS patients. Defects in the cell-mediated immune responses to measles virus have been reported for individuals suffering from MS t Present address: Ben Gurion University of the Negev, Beer-Sheva, Israel.
(26, 27). Recently, measles was reported to be observed by immunofluorescence and by cocultivation in the jejunum of chronic MS patients (21, 22), although this observation has yet to be confirmed. Factors other than an infectious agent appear to be involved in the etiology of MS. Of particular interest is the finding of a marked genetic predisposition to MS, with a high correlation between the incidence of the disease and certain histocompatibility (HLA) types (19). For example, in a study in Western Europe, 70% of MS patients were found to express the HLA specificity, DW2, compared with only 16% of normal individuals (13). One of the problems in understanding the pathogenesis of SSPE and MS has been the lack of suitable animal models. Measles virus is a human pathogen, and it has proven difficult to develop appropriate models for acute and chronic measles infection in laboratory animals. Apart from experiments in nonhuman primates with SSPE (30), most studies have involved inoculation of weanling or neonatal hamsters with wild-type measles (28), SSPE virus (3), or temperature-sensitive mutants of measles (10). The severity of disease observed in these studies was age dependent. Newborn hamsters developed an acute, fatal, giant cell encephalitis, whereas a chronic inclusion cell encephalitis, similar to human SSPE, occurred in weanling animals. The existence of a multitude of inbred strains of mice would enable investigation of any genetic restriction of susceptibility or resistance 764
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SUSCEPTIBILITY OF MICE TO MEASLES INFECTION
to infection with measles. Griffin et al. (7) observed an age-dependent susceptibility to an acute, lethal infection in neonatal BALB/c mice, but did not investigate the influence of host genotype on the outcome of the disease. The present report represents a preliminary study in mice of the genetic basis for susceptibility to the acute, lethal encephalitis which follows intracerebral (i.c.) inoculation of measles virus. In addition, it will be indicated that under appropriate conditions, persistent infection beyond the period of acute infection can be achieved at a low frequency in surviving animals. MATERIALS AND METHODS Mice. Inbred mice of the following strains were used to provide inbred, F, hybrid, and backcross neonates: C3H/HeJ, C3HeB/FeJ, C3H/HeSn, AKR, BALB/c, DBA/2, C57BL/6, C57BL/10 (BO), SJL/J, A/J (all obtained from the Jackson Laboratory, Bar Harbor, Me.) and CBA/Ca (from the Animal Division of the Clinical Research Centre, London, England). In addition, mice of various congenic strains (all obtained from F. Lilly) of BALB/c (BALB.B and BALB.K), of C3H (C3H.Sw), and of C57BL/10 (B1O.Br) were also used in this study. All matings were carried out in this laboratory to provide neonates of the required ages. Virus. The Edmonston strain of measles virus (obtained from B. Fields) was serially passaged in vivo through the brains of neonatal hamsters. After double plaque purification in CV-1 cells, the virus was passed once through neonatal mouse brain, and then virus stocks were prepared by two passages at a low multi-
plicity of infection of 0.01 in CV-1 cells. The virus was harvested by alternately freezing and thawing the infected cell monolyer and medium at 72 h after inoculation, followed by centrifugation at 1,000 x g for 10 min at 40C to remove cell debris. The virus stock, which had a titer of 1.5 x 106 plaque-forming units (PFU) per ml, was stored frozen at -700C until required. Virus titration. Virus samples were titrated by using the semi-microplaque method (23) with CV-1 or Vero cells as indicators. Inoculation of mice. For all experiments, unless otherwise stated in the text, each mouse received an i.c. inoculation of 0.03 ml of undiluted virus stock containing 4.5 X 104 PFU of measles virus. Histology. Tissues were fixed in formal-saline for 48 h, embedded in paraffin wax and microtome sectioned. Sections were stained with hematoxylin and eosin.
Immunofluorescence. Freshly excised tissues were "snap-frozen" in cold (-750C) iso-pentane, cryostat sectioned at 4 to 6 um, and either air-dried or fixed in cold (40C) acetone. Sections were stained with a fluorescein isothiocyanate-conjugated hyperimmune rabbit anti-measles virus serum (hemagglutination inhibition titer, 1/1,280), and rhodamine-labeled bovine serum albumin was used as counterstain. Stained sections were viewed on a Leitz Orthoplan fluorescence microscope with epi-illumination at a wavelength of 490 .m.
Detection of infectious virus in tissues. Brain,
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thymus, liver, spleen, kidneys, and peripheral lymph nodes were removed from acutely infected mice at intervals after inoculation, homogenized with a ground glass homogenizer in 1 ml of Eagle minimum essential medium containing antibiotics and 5% (vol/vol) fetal calf serum, and titrated as described above. To detect the persistence of measles virus, survivors of the acute infection were killed at intervals, and the same organs were removed. Cell suspensions were prepared by mechanical disruption in Eagle minimum essential medium and these were titrated directly; cocultivated with monolayers of Vero indicator cells; or fused with Vero cells in suspension using polyethylene glycol (8), seeded in culture flasks, and cultured until confluent. Cocultivation and fusion cultures were passaged at least twice before discard and were examined daily for measles-induced cytopathic effect. H-2 haplotype determination. The haplotype of backcross progeny mice was determined by a microhemagglutination assay using washed mouse erythrocytes and a specific anti-H-2' immune serum (from D. C. Shreffler).
RESULTS Acute infection. i.c. inoculation of 1- to 2day-old neonatal C3H/HeJ, CBA, and BALB/c mice with measles virus produced an acute lethal disease in all mice. The clinical signs of infection were first apparent 5 to 7 days after inoculation when the mice became hyperactive for about 24 to 48 h. This was followed by a period of ataxia, paralysis, and finally by death. Infected mice exhibited some growth retardation when compared with uninfected, age-matched control mice. Histopathological lesions were observed in the cerebral cortex and hippocampus. Focal lesions of inflammation and necrosis with infiltrating mononuclear cells were frequently surrounded by giant syncytial cells, comprised primarily of neurons. Some focal necrosis of neurons with swelling of nuclei and vacuolation was also observed. The first foci were detected 4 days after inoculation, and the number of lesions increased progressively. After a further 3 to 5 days, the histopathology was severe, and infectious foci were spread throughout the cerebral cortex and hippocampus. Immunofluorescent staining showed that in acutely infected mice, measles viral antigen appeared initially in the cerebral cortex 4 days after inoculation. Infected cells occurred in discrete foci, and the antigen was detected both on the cell membrane and as cytoplasmic inclusions. Nuclear inclusions of viral antigen were also observed in some of these cells. Staining was intense throughout the cerebral cortex 9 days after inoculation, and foci appeared in the hippocampus. Maximum titers of virus were recovered from the brains of infected CBA mice 8 to 10 days after inoculation, which is consistent with the appearance of viral antigen and histopathology (Fig. 1). Similar re-
766
INFECT. IMMUN.
NEIGHBOUR, RAGER-ZISMAN, AND BLOOM
Age- and strain-related susceptibility of inbred mice to acute infection. Mice from eight inbred strains were all found to be highly susceptible to acute infection when inoculated with a high dose of measles (4.5 x 104 PFU) ,D 6D within 2 days of birth (Table 1). However, a variation in susceptibility to virus dose was apparent in some of these strains during the first 2 days after birth when the titer of virus required 'LU 5.0 to produce lethal infection was determined for each strain (Table 2). When mice from some of these strains were inoculated with measles at different ages, there was an age-dependent var4.0iation between strains in susceptibility to infection (Table 3). Although C3H/HeJ mice re2 4 6 8 10 12 14 mained highly susceptible to lethal infection at DAYS AFTER INOCULATION 3 to 4 days of age, BALB/c and C57BL/6 exFIG. 1. Virus titers in brain homogenates at inter- hibited an intermediate susceptibility, and SJL vals after i.c. inoculation of 1- to 2-day-old CBA mice mice were totally resistant. CBA mice and 0
with measles virus. Each point represents the mean titer (± standard error of the mean) from a minimum of three mice on each of the days indicated.
TABLE 1. Mortality rates (%o) in 1- to 2-day-old mice of various inbred strains after i.c. inoculation with measles virus
sults were obtained with C3H/HeJ mice. No infectious virus or viral antigen was detected in the thymus, liver, spleen, kidney, or peripheral lymph nodes of any of the infected mice. To determine the lethal dose of virus for 50% of inoculated mice (LDrA), 1- to 2-day-old CBA mice (14 to 21 mice for each dose) were inoculated i.c. with serial 10-fold dilutions of the stock virus and observed daily for death. Mice which died within 3 days of inoculation were excluded from the data, since death was assumed to have resulted from inoculation trauma. The LD5o was calculated from the mortality rates by the method of Reed and Muench (24), and was found to be 27 PFU per mouse. Inoculation of 1- to 2-day-old mice with measles virus by the intraperitoneal or intranasal routes did not produce any detectable disease or infection.
Strain
No. of mice H-2haplotype inoculated
k k k d d b
C3H/HeJ CBA AKR BALB/c DBA/2 C57BL/6 SJL A/J
s a
15 28 5 10 8 12 8 5
Mortality rate (%)
100 100 100 100 100 91.7 100 100
TABLE 2. LDro of measles virus for 1- to 2-day-old mice of different strains Strain LDr, 10,000
TABLE 3. Mortality rates (%) in neonatal mice of various inbred strains after i.c. inoculation with measles virus at different ages Strain
C3H/HeJ BALB/c
H-2type haplo-
k
1-2a
100
(15/15)b d
100
3-4 100
(19/19) 40.0 (6/15)
5-6
7-8
9-10
11-12
18-20
92.3
85.7
44.4
22.7
0
(48/52)
(12/14)
(4/9)
(5/22)
0
(0/14)
NDc
ND
ND
(0/9) 0 (0/18) ND
0 (0/7 ND
ND
0 (0/6) ND
14.8 (4/27) b 91.7 57.1 0 C57BL/6 (11/12) (8/14) (0/15) s SJL 100 0 0 (8/8) (0/13) (0/15) a Age (days) at inoculation (4.5 x 104 PFU per mouse). ' No. dead/no. inoculated. 'ND, Not done.
(10/10)
Mortality rate (%)
ND
SUSCEPTIBILITY OF MICE TO MEASLES INFECTION
VOL. 21, 1978
other C3H sublines, namely C'3HeB/FeJ and C3H/HeSn, were also found to be highly susceptible (data not shown). In additi( )n, the duration of the acute disease and the day of death varied according to their relative susc eptibilities (Fig. 2). To determine whether the va rying susceptibilities exhibited by these strains were correlated with the extent of viral replicatii on, 5- to 6-day100
C3H
80 60 40 20
_;_S.
'
______t~~~~ 0
LuS
100- C57BI/6 80
60 I.-
40 20 (
100
-
-L
-
-
,
'1I-
SJL
80
60
40 20 0
7
14
21
.1___L.......J 35
28
42
DAYS AFTER INOCLJLATION
FIG. 2. Cumulative mortality raltes in four inbred strains after i.c. inoculation hwith measles virus at I to 2 days (solid line) and 3 to 4 days (dotted line) of age. mouse
767
old C3H, BALB/c, and SJL mice were inoculated with the standard inoculum of measles virus, and their brains were examined for evidence of productive infection at weekly intervals. Table 4 shows that considerable viral replication had occurred in the brains of C3H mice by 7 days after inoculation, there being high titers of infectious virus in most of the brains examined and numerous foci of viral antigen throughout the cerebral cortex. After a further 7 days, no virus was recovered from surviving mice, although a few scattered fluorescent cells were seen in the cerebral cortex. The brains from more resistant BALB/c mice exhibited less evidence for viral infection. Virus was recovered from only one brain, and only a few foci of viral antigen were observed in the cerebral cortex of some mice. By 14 days after inoculation, viral infection appeared as only a few positively stained cells in the Purkinje layer of the cere-
bellum. None of the highly resistant SJL mice showed infection of their brains at any time after inoculation. No virus was recovered, or immunofluorescence observed, in the brains of mice from any of these strains at 21 or 28 days after inoculation. Growth retardation during acute infection. Inoculation of 5- to 6-day-old C3H mice with a lethal dose of measles virus produced a marked retardation in growth before death. This apparent "runting" was found to be associated with thymic dysplasia (Fig. 3). Histological examination of the thymuses from these mice re-
vealed an absence of clear cortical and medullary junctions, and many pyknotic nuclei and karyorrhectic cells were observed. Viral antigen was not detected in these organs by immunofluores-
and infectious virus could not be covered.
cence,
re-
TABLE 4. Production of infectious virus or viral antigen in the brains of C3H, BALBIc, and SJL mice after i.c. inoculation with measles virus Virus antigen
Infectious virus Strain'
after inDays oculation
FrequencyCb
Mean titer (logoPFU/g)
4.3 7 6/7 0 0/4 14 0 0/4 21 0 28 0/2 4.2 7 1/4 BALB/c 0 0/4 14 0 21 0/6 0 28 0/4 0 7 0/7 SJL 0 14 0/7 0 21 0/5 0 0/4 28 a Mice (5 to 6 days old) were inoculated i.c. with 4.5 X ' No. of positive brains/no. examined.
C3H/HeJ
FFrequency'
Location
Numerous foci in cerebral cortex 6/7 Few discrete cells in cerebral cortex 2/4 Negative 0/4 Negative 0/2 Several foci in cerebral cortex 2/4 Few cells in Purkinje layer 4/4 Negative 0/6 Negative 0/4 Negative 0/7 Negative 0/7 Negative 0/5 Negative 0/4 104 PFU of measles virus.
768
INFECTr. IMMUN .
NEIGHBOUR, RAGER-ZISMAN, AND BLOOM
Genetic basis of resistance. Since mice with the H-2k haplotype (C3H) were susceptible to lethal measles infection, and those with the H-2' haplotype (SJL) were resistant, experiments were performed to determine whether the major histocompatibility gene complex controlled host susceptibility to this virus. F1 (C3H x SJL) hybrid mice, 5 to 6 days old, which were heterozygous for the s and k alleles were found to be totally resistant (Table 5). Progeny derived from a backcross of F. (C3H x SJL) hybrids with the susceptible C3H parent exhibited an intermediate susceptibility (56%) when inoculated at 5 to 6 days of age. Surviving mice were typed for their H-2 haplotype, and there was found to be an equal distribution of mice with H-2 /k and H-2k1k (Table 5). Therefore, resistance to acute measles infection appears to segregate independently of the H-2 complex in these two strains. To further investigate the role played by the major histocompatibility gene complex in controlling susceptibility to lethal measles infection, 5- to 6-day-old mice of various congenic strains
were inoculated with this virus. BALB.B (H-2b from C57BL/10) and BALB.K (H-2' from C3Hf/An) did not differ in their susceptibilities from BALB/c (H-2d) (Table 6). However, C3H.Sw (H-2b) mice were significantly more resistant (P < 0.001, chi-square test) than congenic C3H (H-2k) mice, and B10.Br (H-2k) were more susceptible than congenic B10 (H-2b) mice (Table 6). Thus, the inoculation of H-2 congenic mice suggested that in some strains the H-2 complex might modify expression of the gene or genes which determine resistance. Persistent infection. In Table 4 it was indicated that viral antigen could be detected in the brains of acutely infected C3H and BALB/c mice up to 14 days after inoculation. To determine whether neonatal i.c. inoculation with measles virus could lead to a persistent infection, mice which had survived the acute episode were killed at intervals, and their organs were examined for viral antigen by immunofluorescence or infectious virus by cocultivation or cell fusion techniques. Of 18 C3H, 26 BALB/c, 12 C57BL/6, and 18 SJL mice killed between 1 and 14 months after inoculation, viral antigen was found in only 70 3 BALB/c mice, at 58 days after inoculation. In these mice, a few discrete fluorescent cells were 601observed in the cerebral cortex and along the Purkinje layers of the cerebellum. In addition, a 501few fluorescent foci were observed in the spleens of two of the three mice. Fluorescent staining was blocked with unconjugated rabbit anti-meaE 40 sles serum, but not by nonimmune rabbit serum, confirming the specificity of this staining for 030 measles antigen. No infectious virus could be recovered from cells obtained from brain, thy20u-I mus, liver, or spleen of any of the survivors of the acute infection by cocultivation or by poly10 ethylene glycol fusion with permissive Vero cells. 21 3 5 7 10 12 14 17 DISCUSSION DAYS AFTER INOCULATION The results of these experiments show that FIG. 3. Thymic dysplasia after i.c. inoculation of 5- to 6-day-old C3H mice with measles virus: unin- the Edmonston strain of measles virus is neurofected mice (0); infected mice (0). virulent for newborn mice. i.c. inoculation during uJ
-
TABLE 5. Relationship between H-2 and susceptibility to lethal measles infection No. of
Strain" Strain'
iicN in miocelam-
H-2 haplotype frequency' of inMortality rate
C3H SJL
(%
oculated mice
s/s
52 92.3 0 15 0.0 100 18 0.0 0 F,(C3H x SJL) 0 C3H x F. (C3H x SJL) 74 56.8 aMice (5 to 6 days old) were inoculated i.c. with 4.5 x bAll frequencies are theoretical unless indicated. Frequency determined by sera typing. d
Frequency calculated by subtraction.
s/k
k/k
H-2 haplotype frequency of surviving mice
s/s
s/k
k/k
0 100 0 0 0 100 100 0 0 0 51c 49d 104 PFU of measles virus.
0 0 100
100 0 0
54C
46d
VOL. 21, 1978
SUSCEPTIBILITY OF MICE TO MEASLES INFECTION
TABLE 6. Susceptibility of various congenic strains to lethal measles infection Strain'
H-2type haplo-
No. of mice inoculated
Mortality (%) rate
BALB/c BALB.B BALB.K C3H C3H.Sw B10
d 27 14.8 b 23 17.4 k 12 16.7 k 52 92.3 b 20 40.0 b 19 47.4 B10.Br k 70 70.0 a Mice (5 to 6 days old) were inoculated i.c. with 4.5 x 104 PFU of measles virus.
the first 48 h after birth produced an acute, lethal encephalitis in almost all mice. Many mice were runted, and this was probably a result of the thymic dysplasia which occurred in these mice. There was an age-related development of resistance to infection, and the rate of this development appeared to be strain dependent. The various inbred strains of mice used in this study could be designated as being either susceptible, resistant, or intermediate when inoculated after 2 days of age. However, even in the highly susceptible strains, the period of susceptibility was short-lived. The consequence of acute infection was clearly related to the extent of virus growth in the brain as shown by the recovery of infectious virus and by the appearance of viral antigen at intervals after inoculation. Susceptible C3H mice exhibited numerous foci of infection in the cerebral cortex, and virus was recovered from almost all of the mice inoculated. The extent of the infection was somewhat reduced in a strain of intermediate susceptibility, namely BALB/c, whereas totally resistant SJL mice failed to exhibit any viral growth in brain tissues. It is not known whether this reduction in virus growth results from increased immune responsiveness or whether it reflects different patterns of neurodifferentiation with loss of measles-permissive cells in resistant mice. Differences in susceptibility in various strains of inbred mice suggested that the major histocompatibility complex might be implicated in the control of susceptibility or resistance to acute measles infection. It is known, for example, that resistance to murine leukemia is controlled by a number of genetic loci, two of which, Rgv-1 (15) and Rfv-1 (4), are associated with H2. Rgv-1 maps proximally towards the K region of H-2 (15), and it has been suggested that this gene might be linked to the immune response (Ir) locus (18). Indeed, if the immune response is responsible for recovery from acute measles infection in resistant mice, an appropriate Ir-
769
associated gene might determine susceptibility or resistance to this virus. This is believed to be the case in mice for susceptibility to lymphocytic choriomeningitis virus disease (20). However, in a number of other systems, the major determinants of resistance are not associated with the major histocompatibility complex. For example, the two major determinants of susceptibility to Friend virus-induced leukemogenesis, called Fv-l and Fv-2, map on chromosomes 4 and 9, respectively (25). Resistance of mice to various myxoviruses is determined by a single dominant autosomal locus called Mx (16), and three nonH-2-linked genes are believed to control resistance of mice to herpes simplex virus infection (17). In the present study, the gene or genes determining resistance to measles were found to be dominant, since F. hybrids derived from susceptible (C3H) and resistant (SJL) parental strains were totally resistant to lethal infection. However, backcrossing of these resistant F, mice with the susceptible parental strain (C3H) showed that resistance segregated independently of H-2. Although the simplest interpretation of these data would suggest that a single, dominant, nonH-2-linked gene determines susceptibility or resistance, further testing of progeny from these F2 mice for their susceptibility to acute infection will be required to estimate the number of genes involved. Data obtained from the inoculation of certain congenic strains suggests that although the H-2 complex does not determine susceptibility or resistance, it might modify expression of the gene or genes involved. C3H.Sw mice, which were significantly more resistant than congenic C3H mice, have a resistant H-2 haplotype (H-28) on a susceptible C3H background. Conversely, B1O.Br were more susceptible than congenic B10 mice, and they have a susceptible H-2 haplotype (H-2k) on a resistant background. BALB/c congenic strains did not exhibit any significant changes in susceptibility despite their different H-2 haplotypes. Finally, because two slow neurological diseases of humans, SSPE and MS, have been associated with persistent measles infection, an attempt was made to determine whether Edmonston measles virus could persist in mice. A few survivors of acute infection did exhibit persistence of measles virus antigens in cells of the cerebellum and spleen, but the frequency was very low (3/74). We must conclude that, at the present time, i.c. inoculation of neonatal mice with this strain of measles virus constitutes an unsatisfactory model for the investigation of persistent measles infection. Perhaps with adaptation of this virus to murine nervous tissues,
770
NEIGHBOUR, RAGER-ZISMAN, AND BLOOM
immunosuppression, or passive transfer of measles antibody, the model could become more useful for the investigation of persistent measles infection. ACKNOWLEDGMENTS This study was supported by National Multiple Sclerosis Society grant 1006-B-2. We are grateful to Grace Ju for helpful discussions in preparation of this manuscript and to R. D. Barnes, Clinical Research Centre, London, England, for providing facilities for part of this investigation.
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ence to clinical course. Lancet ii:1221-1224. 14. Johnson, R. T. 1975. The possible viral etiology of multiple sclerosis. Adv. Neurol. 13:1-46. 15. Lilly, F. 1970. The role of genetics in Gross virus leukemogenesis, p. 213-220. In R. M. Dutcher (ed.), Comparative leukemia research, 1969. Karger, Basel. 16. Lindenmann, J. 1964. Inheritance of resistance to influenza virus in mice. Proc. Soc. Exp. Biol. Med. 116:506-509. 17. Lopez, C. 1975. Genetics of natural resistance to herpesvirus infections in mice. Nature (London) 258:152-153. 18. McDevitt, H. O., M. B. A. Oldstone, and T. Pincus. 1974. Histocompatibility-linked genetic control of specific immune responses to viral infection. Transplant. Rev. 19:209-225. 19. McFarlin, D. E., and H. F. McFarland. 1976. Histocompatibility studies and multiple sclerosis. Arch. Neurol. 33:395-398. 20. Oldstone, M. B. A., F. J. Dixon, G. F. Mitchell, and H. 0. McDevitt. 1973. Histocompatibility-linked genetic control of disease susceptibility. J. Exp. Med. 137:1201-1212. 21. Pertschuk, L. P., A. W. Cook, and J. Gupta. 1976. Measles antigen in multiple sclerosis: identification in the jejunum by immunofluorescence. Life Sci. 19:1603-1608. 22. Prasad, I., J. D. Broome, L. P. Pertschuk, J. Gupta, and A. W. Cook. 1977. Recovery of paramyxovirus from the jejunum of patients with multiple sclerosis. Lancet i:1117-1123. 23. Rager-Zisman, B., and T. C. Merigan. 1973. A useful quantitative semimicromethod for viral plaque assay. Proc. Soc. Exp. Biol. Med. 142:1174-1179. 24. Reed, L. J., and H. Muench. 1938. A simple method of estimating fifty percent end points. Am. J. Hyg. 27:493-497. 25. Steeves, R., and F. Lilly. 1977. Interactions between host and viral genomes in mouse leukemia. Annu. Rev. Genet. 11:277-296. 26. Utermohlen, V., J. Farmer, J. Kornbluth, and M. Kornstein. 1977. The relationship between direct migration inhibition with measles antigen and E rosettes in normals and patients with multiple sclerosis. Clin. Immunol. Immunopathol. 9:63-66. 27. Utermohlen, V., and J. B. Zabriskie. 1973. A suppression of cellular immunity in patients with multiple sclerosis. J. Exp. Med. 138:1591-1596. 28. Wear, D. J., and F. Rapp. 1971. Latent measles virus infection of the hamster central nervous system. J. Immunol. 107:1593-1598. 29. Wechsler, S. L., and B. N. Fields. 1978. Differences between the intracellular polypeptides of measles and subacute sclerosing panencephalitis virus. Nature (London) 272:458-460. 30. Yamanouchi, K., N. Uchida, S. Katow, T. A. Sato, K. Kobune, F. Kobune, Y. Yoshikawa, H. Kodama, and A. Shishido. 1976. Growth of measles virus in nervous tissues. IV. Neurovimlence in wild measles and SSPE viruses in monkeys. Jpn. J. Med. Sci. Biol. 29:177-186.
