The Institute for Infectious Diseases, The University of Tokyo, Minato-ku, Tokyo, Japan
A Neurotropie Variant o[ Measles Virus in Suckling Mice By
Minoru Matumoto*, Yasuo Saburi, Yuzo Aoyama, and Masahiko Mutai With 8 Figures
(Received .February 29, 1964) Since the successful propagation of measles virus in tissue cultures of human and simian kidneys by Enders and Peebles (2), renewed attempts have been made at transmitting the virus to mice. Imagawa and Adams (3) reported serial intracerebral passage in suckling mice of the Edmonston strain adapted to HeLa cells. GarlstrSm (1) succeeded in adapting a strain, passaged 11 times in human renal cell culture, to suckling mice by the same route of inoculation. Walcsman et al. (7) reported serial intracerebral passage in suckling hamsters of the Philadelphia 26 strain and its adaptation with continued passage to the brains of older hamsters and mice. The present report is to describe our experiences with the Sugiyama strain of measles virus which has been finally adapted to the brain of suckling mice after numerous passages in cell cultures. The observations of the mouse passage, the behaviour in mice of the mouse adapted virus and the histologie and fluorescent antibody studies of infected mice are presented.
Materials and Methods Virus: The Sugiyama strain (5) was employed in most of the experiments but the Tanabe, Tanaka and Edmonston strains were also used. The strains, except the Edmonston strain which was originally isolated by Euders and Peebles (2), were isolated in monkey renal cell cultures from throat swabs of measles patients in Tokyo. They were used in the present study after passages through various cell cultures and or developing chicken eggs as indicated in * The Institute for Infectious Diseases, The University of Tokyo, Minato-ku, Tokyo, Japan.
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iV[. Matumoto, u
Saburi, Y. Aoyama, and M. Mural:
Table 1. Superuatant fluids from frozen and thawed, infected cultures or 10% suspensions of infected amniotie membranes of chick embryos were used as inoeulum for the initial passage in suckling mice.
Mouse inocuZation: Laboratory-bred litters of albino mice, dd line, were inoculated intracerebrally within 24 hours after birth with 0.01 ml. of inoeulum. For the passage, a 20% suspension of infected brains was prepared in YLE medium (Earle's solution containing 0.5% lactalbumin hydrolysate, 0.1% yeast extract, 500 units per ml. of penicillin, and 100 ttg per ml. of streptomycin) supplemented with 5% bovine serum. The suspension was centrifuged at 3000 r. p. m. for 10 minutes and the resulting supernatant fluid was used for intracerebral inoculation of mice. Virus titration: Virus was titrated in tube cultures of human amniotic F L cells (5). Supernatant fluids from infected cultures or 20% suspensions of infected mouse brains were diluted serially (10-fold per step) in Y LE medium and each dilution was inoculated in 0.1 ml. amounts into 3 tube cell cultures. After adding 0.9 ml. of YLE medium supplemented with 5% bovine serum, the inoculated cultures were incubated in a roller drum at 37 ~ C for 7 days. The TCID~0 titer was calculated on the basis of cytopathic effect by Kdirber's method (4). The mouse LDs0 titer was determined by injecting each of 10-fold serial dilutions intracerebrally into 3 suckling mice. Neutralization test: A 1 : I mixture of diluted serum, heated at 56~ for 30 minutes, and diluted virus, was held at room temperature for 30 minutes and then at 4 ~ C for 30 minutes, or at room temperature for 90 minutes, before inoculation of four tube cultures of F L cells in 0.2 ml. amounts. The inoculated cultures were incubated in a roller drum at 37 ~ C for 10 days and the tubes showing no eytopathic effect were taken as positive. In the neutralization test in suckling mice, the virus-serum mixture prepared according to the above procedure was injected intracerebrally in 0.01 ml. amounts into suckling mice, which were clinically observed for 3 weeks. Fluorescent antibody technique: The direct method was employed. The fluorescent antibody employed in the present study was prepared by Drs. A. Kawamura and H. Nagahama with convalescent serum from a Japanese monkey infected subcutaneously with the Sugiyama strain grown in F L cell cultures. The immune serum used had a complement fixing titer of 1/256 and a neutralizing titer of 1/29.5 with 100 TCID~0 of virus. Globulin was precipitated from the serum with half.saturated ammonium sulfate at 2 ~ C and then three times with one third-saturated ammonium sulfate. The resulting globulin fraction dissolved in 0.15 M NaC1 solution was dialysed with stirring overnight against 0.15 M NaC1 solution. The protein content of the solution obtained was 1.8%. Fluorescein isothioeyanate (Baltimore Biochemical Lab., Inc.), 1/100 of the globulin by weight, was dissolved in 0.5 M carbonate-bicarbonate buffer solution, pH 9.5, 1/10 in volume of the globulin solution employed. The globulin solution and the dye solution were mixed and incubated with stirring at 4 ~ C for 4 hours. The mixture was then subjected to chromatography on a Sephadex G25 column with 0.005 IV[ phosphate buffer in 0.1 NaC1, pH 7.0. The conjugated fraction obtained underwent D E A F column chromatography with the same buffered saline as used above and the fractions with fluorescein-protein molecular ratio of 1.0 to 2.0 were collected. The staining activity of the preparation obtained was shown, at a dilution of up to 1 : 16, when tested with F L and Hep-2 cell cultures infected with the Sugiyama strain. According to this finding the conjugated globulin solution was diluted four-
A Neurotropie Variant of Measles Virus in Suckling Mice
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fold with a solution of 0.005 M phosphate buffer and 0.15 M NaC1, pH 7.0, and sufficiently absorbed with acetone powders of human liver and brain and mouse liver to remove non-specific staining activity and was ready for use.
The specificity of the conjugated globulin was carefully tested with FL cells both normal and infected with the Sngiyama strain and with sections of mice both normal and infected with herpes and Japanese encephalitis viruses. The tissue fragment to be examined was quickly frozen in hexan chilled in acetone-dry ice mixture and thin sections, 2 S thick, were made in a Pearse SLEE cryostat. Sections mounted on cover slips were fixed with acetone at --20 ~ C for one hour and, after drying, stained with fluorescent antibody at 5~ C for 15 hours. The stained preparations were rinsed with phosphate buffered saline, dried, and mounted on slide glasses with carbonate-bicarbonate buffered glycerine solution, pH 7.2, for microscopy. The stained sections were examined under dark-field illumination using a high-pressure mercury-vapor lamp, H-250 (Chiyoda Optical Co.) as UV source and UV exciter filter and UV absorbing filter (Chiyoda).
Results
Occurrence o/variant strain Attempts were made at infecting suckling mice by the intracerebral route with the Sugiyama strain at various passage levels. The results are summarized in Table 1. The first trial was made with virus MK6 which had undergone 6 passages in monkey kidney cell cultures after isolation. Neither illness nor multiplication of virus occurred in the inoculated mice even though 4 additional blind passages were made. Virus MK6 was further passaged 4 times in cultures of human conjunctival cells of Chang and then adapted to bovine renal cell cultures (6). The virus at the 13th and 50th passages in bovine kidney cells (MK6-Con4-BK13, MK6-Con4-BK50) failed to infect suckling mice. Three blind passages from the mice inoculated with the 13th passage material also gave negative results. I n another passage series the Sugiyama strain was adapted to chick embryos by the amniotic route of inoculation after 6 passages in monkey kidney cell cultures and 6 passages in conjunctival cell cultures of Chang. After 36 passages in developing eggs the virus was passed once in a F L cell culture (MK6Con6-DEA36-FL 1) and inoculated into the brain of suckling mice with negative results. Virus MK6 of the Sugiyama strain was passaged through F L cell cultures and tested at various passage levels. The virus materials at the 10th (MK6-FL10), 13th (MK6-FL13) and 49th (MK6-FL49) passage levels failed to infect suckling mice. With virus MK6-FL13 four blind passages were made with negative results. I n contrast with those earlier passage materials the virus at the 76th F L passage (M_K6-FL76) produced illness in 6 of 7 inoculated mice. Likewise, suckling mice inoculated with the later passage materials, MK6-
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M. Matumoto, Y. Saburi, Y. Aoyama, and M. Mural:
Table 1. A t t e m p t s
Strain
Sugiyama
Tanabe Tanaka Edmonston
at infecting suckling mice with measles virus by intraeerebral route Passage history 1
V i r u s 4ose p e r m o u s e 2 l o g (TCID~0)
Infection rate 8
MK6 MK 6-Con4-BK 13 MK 6-Con 4-B K 50 MK 6-Con 6D E A 36-FL 1 MK 6-FL 10 MK 6-FL 13 MK 6-FL 49
2.0 2.5 n.t.
0/84 0/105 0/S
1.2 3.8 4.5 2.5
0/s 0/56
MK 6-FL 76 MK 6-FL 125 MK 6-FL 126 MK 6-FL 124DEA 1
2.5 2.5 3.5
6/7
1.75
6/s
MK 1-HeLa 3-FL2 MK 1-FL 14 H K 1-HA28)5K 1-FL 6
2.5 n.t.
0/4
2.8
0/10
0/6 0/me 3/4 5/8
0/8
1 P r i m a r y cultures : MK = monkey kidney, B K = bovine kidney, H K = human kidney, HA = human amnion. Stable cell cultures: Con = human conjunctival cell, F L = human amniotic cell, HeLa = human cancer cell. D E A : passage in developing chicken egg b y the amniotic route. The figures represent the number of passages in the indicated host. Determined in F L cell culture. 3 Number of mice either dead of infection or sacrificed when moribund over total number of inoculated mice. 4, 5, 6 Additional blind passages were carried out 4, 3 and 4 times, respectively, with negative results. n . t . : not tested. F L 1 2 5 , 126, M K 6 - F L 1 2 4 - D E A 1 , also b e c a m e ill w i t h a m o r t a l i t y of 3/4, 5/8, a n d 6/8, respectively. T h e change in p a t h o g e n i c i t y o b v i o u s l y occurred somewhere b e t w e e n t h e 4 9 t h a n d 76th passage in F L cells. T h e i n c u b a t i o n p e r i o d was 9 to 13 days, 10 d a y s being t h e m o s t common. T h e earliest m a n i f e s t a t i o n of illness was i r r i t a b i l i t y : affected animals r a n a n d j u m p e d in t h e cage when d i s t u r b e d . S u b s e q u e n t l y , t h e y b e c a m e a p a t h e t i c a n d d e v e l o p e d spastic p a r a l y s i s of limbs w i t h disequilibrium, r i g i d i t y a n d m u s c u l a r twitching. D e a t h g e n e r a l l y occurred 3 to 5 d a y s a f t e r initial signs of illness. The T a n a b e , T a n a k a a n d E d m o n s t o n strains a t certain p a s s a g e levels, as shown in T a b l e 1, were also t e s t e d for i n f e c t i v i t y in suckling mice a l t h o u g h w i t h n e g a t i v e results.
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Serial passage o/the variant strain in suelcling mice Virus M K 6 - F L 1 2 4 - D E A 1 with a t i t e r of ]0 +=.75TCIDs0 per 0.1 ml. was used as i n o c u l u m of the i n i t i a l passage (see T a b l e 1). The results of the serial passage are s u m m a r i z e d in Table 2. The first 4 passages were readily carried out, a l t h o u g h some of the i n o c u l a t e d mice showed no signs of illness, the infection rate being 56% of the 25 i n o c u l a t e d mice. The virus titer of the b r a i n suspension was geTable 2. I n t r a c e r e b r a l
Passage 13
2 3 4 5
No,
passage
Infection rate 1
6/8 1/5 4/8 3/4 O/7
of S u g i y a m a mice Incubation
strain
(days)
11 12 11-13 9--11
in suckling
V i r u s t i t e r of b r a i n ~ log (TCIDso/g)
2.7 3.2 3.2 2.2 negative
(discontinued) 54
7 9 10 13 16 17
6/6 2/7 6/6
lO/lO 9/9 5/5 6/7
7--8 12 5--8 6--8 5--8 8 8--9
n.t. 3.9 6.2 4.7 n.t. n.t.
1 2 See the foot-notes in Table 1. a The virus MK6-FL124-DEA1 (see Table 1) was used as inoculum of the initial mouse passage. 4 Since the passage failed at the 5th generation, the virus harvested from the mouse brains at the 4th passage was grown in FL cell culture and the resulting infectious culture fluid was used for the further mouse passages. n . t . : not tested. n e r a l l y low. The b r a i n suspension used for the 5th passage contained o n l y 100.5 TCIDs0 per 0.1 ml. All of the 7 i n o c u l a t e d mice at this passage failed to develop clinical signs of illness, a n d no virus was recovered from b r a i n tissues of sacrificed mice. Hence, the b r a i n suspension harvested at the 4th mouse passage was i n o c u l a t e d once into F L cell cultures a n d the resulting virus was used to c o n t i n u e mouse passages, which were readily accomplished u p to the 17th passage, where the serial passage was discontinued. The i n c u b a t i o n period was 9 to 13 days at the early passages a n d became shorter, 5 to 9 days, from the 9th passage on. The infection rate, as m e n t i o n e d above, was a b o u t 50~o t h r o u g h the first 4 passages b u t almost
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100% at t h e 9 t h a n d l a t e r passages. The clinically affected mice u s u a l l y died 3 to 5 d a y s after i n i t i a l signs of illness, b u t a few of t h e m recovered. The virus t i t e r of infected mouse brains, as d e t e r m i n e d in F L cell cultures, was low, ranging from 10 ~.~ to 103'2 TCIDs0 p e r g r a m at t h e first 4 passages, a n d b e c a m e s o m e w h a t higher, 103.9 to 10 G-2, after t h e 9th passage. Table 3. P a r a l l e l t i t r a t i o n s of t h e m o u s e - a d a p t e d Sugiyama s t r a i n b y c y t o p a t h i e e f f e c t in F L c e l l c u l t u r e s a n d i n t r a c e r e b r a l i n o c u l a t i o n of s u c k l i n g m i c e V i r u s d i l u t i o n (-log)
No, positives / T o t a l No. i n o c u l a t e 4 5$ouse (0 01 m].)
0
9/9
1
7/9 s/lo 2/7 o/6 o/3
2 3 4 5 LDso or TCIDs0/0.1 ml.
10a.4
F L (0.1 ml.)
3/3 2/3
1/3 o/3 1Oa.5
The virus material: 20% suspension of infected mouse brains at the 13th mouse passage. Table 4. M u l t i p l i c a t i o n of t h e m o u s e - a d a p t e d i n t h e b r a i n of s u c k l i n g m i c e
Sugiyama
strain
VirtL,~ t i t e r of b r a i n log (TCID~o/g) Days after inoculation Experiment I
3 5 6
7 8
lO
2.95 3.2 3.45* 5.2* 5.95* 5.2*
Experiment II
2.2 4.2* 4.7 3.7 4.45* 5.95*
Inoculum : 0.01 ml. containing 101.~ TCIDs0 of the virus from the 9th mouse passage. The virus titer was determined in tube cultures of F L cells with a pooled suspension of 2 or 3 brains. When a brain or brains from paralytic mouse were present among the brains for infectivity titration the titer obtained is indicated b y an asterisk. The 2 0 % b r a i n suspension from t h e 13th passage was t i t r a t e d in b o t h F L cell cultures a n d suckling mice. The F L t i t e r was l03A TCIDso p e r 0.1 ml., p r a c t i c a l l y equal to t h e mouse i n t r a c e r e b r a l t i t e r of 103.5 LDs0 p e r 0.1 ml. (Table 3).
Multiplication o] the mouse-adapted virus in suckling mice A n u m b e r of suckling mice were i n o c u l a t e d i n t r a c e r e b r a l l y w i t h i n 24 hours after b i r t h w i t h t h e 2 0 % suspension of i n f e c t e d b r a i n s a t t h e
A Neurotropie Variant of Measles Virus in Suckling Mice
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9th mouse passage. Each mouse received 0.01 ml. containing 101"~TCIDh0 of virus. At intervals 2 or 3 mice were taken at random and sacrificed to harvest the brains for infectivity assay in F L cell cultures. Two independent experiments were performed. The results obtained are summarized in Table 4. The virus began to increase about 3 days after inoculation and reached a peak titer of 105~6 TCIDho per gram of brain tissue in 5 to 7 days. Virus was undetectable in various organs other than the brain in infected mice. Thus, 5 suckling mice were sacrificed 7 days after intraeerebral inoculation with 0.01 ml. containing 102"5 TCIDho of virus which had undergone 17 mouse passages plus one F L cell passage, and a Table 5. S u s c e p t i b i l i t y of s u c k l i n g mice of d i f f e r e n t ages to t h e 10% suspension of the heart, lungs, liver, spleen, kidneys and m o u s e - a d a p t e d S u g i y a m a s t r a i n spinal cord of each mouse was Age in days Infection rate tested for infectivity in F L cell 9/9 cultures. The results were invariably negative, whereas the brains 7/7 of those mice contained virus in 6/9, 0/9 0/7, 0/3, 0/9* a titer of 104.3 TCIDao per gram.
o/8, 1/4 Susceptibility of suckling mice o/ di]ferent ages to the mouse-adapted virus
o/6 Inoculum: 0.01 ml. containing 103.5 TCIDh0 of the virus from the 12th mouse passage. The infection rate with an asterisk was obtained with the virus st the l l t h mouse passage, the dose per mouse being 10~.s TCID~0.
Suckling mice, one to 9 days of age, were inoculated intracerebrally with 0.01 ml. amounts, each receiving 102"~TCID~0 of virus at the 12th mouse passage. In one experiment with 7-day-old mice the virus material of the l l t h mouse passage was used with a dose of 102.s TCIDh0 per mouse. The results are shown in Table 5. Mice under 5 days of age were highly susceptible to the virus : almost all the mice inoculated succumbed following infection. With 6-day-old mice death following infection occurred in 6 of 9 inoculated mice in one experiment, but in none of 9 inoculated mice in another experiment. Mice aged 7 days or over failed to be infected except one 8-day-old mouse.
Extraneural inoculation o/suckling mice with the mouse-adapted virus The F L passage virus MK6-FL 126 (104.5 TCIDho/0.1 ml.) and the virus from the 9th mouse passage (10a.~TCIDho/0.1 ml.) were inoculated into dayold suckling mice by the intracerebral and intraperitoneal routes of inoculation with 0.01 ml. and 0.1 ml. amounts, respectively. Death following infection occurred by intracerebral inoculation in 5 of 8 mice inoculated with the
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A Neurotropic Variant of Measles Virus in Suckling Mice
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F L passage virus and in 6 of 6 mice, inoculated with the mouse passage virus, whereas death occurred in none of 5 and 6 mice inoculated intraperitoneally with the F L a n d mouse passage virus, respectively. Intranasal inoculation with the virus did not produce any signs of illness in suckling mice.
Mouse-adapted virus in FL cells
As mentioned above, the virus undergoing intracerebral serial passage in suckling mice proliferated well in F L cell cultures and a cytopathic effect was readily produced similar to that of the original F L passage virus. Serial passage of the mouse-adapted virus was readily accomplished in F L cell cultures by inoculating supernatant fluids from infected cultures.
Identi/ication o/the mouse-adapted virus by neutralization test The mouse-adapted virus at the 8th passage was tested by neutralization in F L cell cultures and in one day-old mice by intracerebral inoculation. The immune serum employed was prepared in a/monkey by subcutaneous inoculation with virus MK6-Con6-BK34 of the Sugiyama strain. The immune serum completely inhibited cytopathic effects in F L cell cultures when tested undiluted with a 20% infected mouse brain suspension. The control series with f0-fold serial dilutions of the same virus suspension and normal bovine serum showed a infectivity titer of 102.75 TCIDs0 per 0.1 m]. The virus-immune serum mixture employed in the above experiment was inoculated intracerebrally into 7 suckling mice, 24 hours of age, and none died of infection. Another group of 7 mice of the same age was inoculated intracerebra]ly with the virus suspension mixed with an equal volume of normal bovine serum and all succumbed with the typical signs of illness. The mouse-passage virus was tested once more by the serum dilution method of neutralization test in F L cell cultures. The virus harvested from mice at the 17th passage was passaged twice in F L cell cultures and the resulting virus material was used for the test. As the control virus, virus M K 6 - F L l l of the Sugiyama strain was used. The antiserum employed was prepared in guinea pigs by 4 subcutaneous inoculations, one week apart, with 1.0 ml. each of the Edmonston strain grown in F L cell cultures and concentrated 10 times by centrifugation. The serum was taken one week after the last injection. The serum was diluted 50-fold and heated at 56 ~ C for 30 minutes before being used for the test. Each of the virus P l a t e I : Fig. 1. M a n y n e r v e cells with s t r o n g l y cosinophilic c y t o p l a s m a n d p y k n o t i e nucleus in t h e c e r e b r a l cortex, 9 d a y s a f t e r infection. H. E. staining. Fig. 2. M a ] t i n u c l e a t e d g i a n t cells in the p y r a m i d a l cell l a y e r of t h e A m m o n ' s horn, 9 days a f t e r infection. G a l l o c y a n i n staining. Fig. 3. Slight p e r i v a s e u l a r cuffing a n d glia cell proliferation in the cerebral w h i t e m a t t e r , 9 days a f t e r infection. H. E. staining. Fig. 4. Specific fluorescence in t h e c y t o p l a s m of n e r v e cells in the cerebral cortex, corres p o n d i n g to P l a t e I, 1. F l u o r e s c e n t a n t i b o d y staining.
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A Neurotropic Variant of Measles Virus in Suckling Mice
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preparations was diluted so as to contain 100 TCID~0 per 0.1 ml. and mixed with each of 2-fold serial dilutions of the serum. The initial serum dilution at the 5 0 % end-point of neutralization, as calculated by the Ki~rber method (5), was 1/100• for the control virus and 1/100• for the mouse-adapted virus.
Histologic observations and development o] speci/ic measles antigens in in/ected mouse brain The histologic changes in the brain of infected mouse were degeneration of nerve cells (Plate I, 1), formation of multinuclear giant cells (Plate I, 2), and proliferation of glia cells (Plate I, 3). These changes were observed diffusely throughout the brain with little tendency to localize in particular areas. No changes were recognized in the cerebellum, spinal cord and visceral organs. The multinueleated giant cells were considered to be formed by fusion of nerve cells and were most frequent in the Ammon's horn. Meningitis and perivascular cuffing (Plate I, 3) were slight. Cytoplasmic inclusion bodies characteristic of measles virus were occasionally found in nerve cells. In the fluorescent antibody study specific fluorescence was first detected in nerve cells in the vicinity of the inoculation site one day after inoculation. Four or five days after inoculation specific fluorescence began to appear in the cortex of the opposite hemisphere and fluorescent cells increased in number with time. By the time of the initial signs of illness fluorescent cells could be found throughout the brain, i.e. in the mantle cortex (Plate I, 4), Ammon's horn (Plate II, 5), and nuclei in the brain stem (Plate II, 8), not localized in purticular areas of the brain. Ammon's horn, however, always exhibited specific fluorescence even in cases not much affected. No specific fluorescence was demonstrated in the cerebellum, medulla oblongata and spinal cord. Specific fluorescence was recognized in nerve cells, rarely in glia cells, too, but never in endothelial cells of blood vessels, ependymal cells of the cerebral ventricles, and cells in the choroid plexuses and the meninges. Specific antigens detectable by fluorescent antibody were always in the cytoplasm, never in the nucleus. Specific fluorescence in the cytoplasm occurred in masses of irregular shape (Plate II, 6, 8) or in fine granules (Plate I, 4; Plate II, 5). In the cytoplasm of multinucleated giant cells fluorescent materials could be found (Plate II, 5, 7). Plate I I : Fig. 5. ,~r g i a n t cells s h o w i n g specific fluorescence in t h e A m m o n ' s horn, corresponding to P l a t e I, 2. F l u o r e s c e n t a n t i b o d y staining. Fig. 6. N e r v e cells exhibiting specific fluorescence in i r r e g u l a r masses and fine granules in the cerebral cortex, 11 d a y s a f t e r infection. F l u o r e s c e n t a n t i b o d y staining. Fig. 7. G i a n t cells w i t h specific fluorescence in the olfactory br~in, 11 days a f t e r infection. F l u o r e s c e n t a n t i b o d y staining. Fig. 8. Specific fluorescence occm'ring in large irregular masses in the b r a i n s~em, 11 days a f t e r infection. F l u o r e s c e n t a n t i b o d y staining.
694
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No specific fluorescence was demonstrated in any tissues other than the brain in infected mice. In accordance with the absence of clinical and virological evidence for infection, no specific fluorescence was shown in the brain of mice inoculated by the intraperitoneal or intranasal route.
Discussion The clinical observations, the infectivity titer o2 mouse brain at each passage, the growth curve of the virus in mouse brain, and the results of the histologic and fluorescent antibody studies all indicate the propagation of the Sugiyama strain of measles virus in intracerebral serial passage in one day-old mice. The neutralization tests and the fluorescent antibody studies of infected mouse brain give definite evidence for the identification of the mouse-adapted virus as measles virus. One of the findings of interest in the present study is that the Sugiyama strain acquired the ability to propagate in the brain of b a b y mice somewhere between the 49th and 76th passage in F L cell culture. Repeated attempts at infecting one day-old mice b y the intracerebral route of inoculation with the Sugiyama strain failed at the earlier passages in F L cells and with virus of various other passage histories, while the virus at the subsequent passages in F L cells infected b a b y mice (Table 1). In contrast with this finding, CarlstrSm (1) was successful in propagating measles virus, passuged only 11 times in human renal cell culture since its isolation, in intracerebral serial passage in one day-old mice. The exact passage history of the Edmonston strain, which Imagawa and Adams (3) used as inoculum for initial mouse passage, was not given in their report but the strain seems to have had numerous passages in human tissue cultures and was finally adapted to H e L a cells. As to the hamster adapted virus of Walesman et el. (7), mice were fully susceptible at all ages to late passage virus, while resistant to early passage virus. A few attempts with the Edmonston and other strains of measles virus in the present study failed to infect b a b y mice. An increased adaptation of the virus occurred to some extent during the serial mouse passage, as evidenced by some increase in the mortality rate and the infective titer of mouse brain and by shortening of the incubation period. Similar observations were made bylmagawa andAdams (3) and CarlstrSm (1). From the 9th passage on almost 100% of the inoculated baby mice developed a lethal spastic paralysis. The clinical manifestations of infected mice were essentially the same as described by Imagawa and Adams (3), Carlstr6m (1) and Waksman et el. (7). The age of the mouse and the route of inoculation are the factors of importance involved in the susceptibility of the mouse to the virus. Mice, 5 days of age or younger, died of infection in nearly 100%, but almost 100% of mice, 7 days o2 age or older, failed to develop any signs of illness.
A Neurotropic Variant of Measles Virus in Suckling Mice
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In contrast with this, the virus of Walcsman et al. (7) infected mice at all ages. The intraperitoneal and intranasal inoculation of day-old mice with our mouse virus produced neither clinical manifestations nor evidence for infection of the virus. The mouse virus multiplied in F L cell culture and readily produced cytopathic effect like the original FL passage virus. Serial passage in F L cell culture of the mouse virus was readily accomplished b y inoculating supernatant fluids from infected cultures. This finding is at variance with t h a t of Imagawa and Adams (3), who reported that, after the 4th mouse passage, serial transfer of their mouse-adapted measles virus in H e L a cell culture was difficult when supernatant fluids were employed, whereas cytopathogenesis was readily induced when infected cells were used as inoculum. I n mice intracerebrally infected with the mouse adapted virus, no evidence for infection in the extraneural tissues was obtained. Furthermore, the virus activity seems to be confined to the cerebrum, not involving the cerebellum, medulla oblongata, and spinal cord, as evidenced b y histologic examination and fluorescent antibody study. A striking and characteristic feature of the disease in suckling mice by this agent is the rather remarkable specificity of the nerve cell involvement. The infected cells showed degenerative process with or without formation of inclusion bodies, and characteristic multinuclear giant cells were formed by fusion of nerve cells. Cytoplasmic inclusions were readily found in occasional nerve cells but as to intranuclear inclusions further examination is necessary to be definite, although some likely looking bodies were found. Walcsman et al. (7) could not detect multinuclear giant cells in mice, but in hamsters. With our virus, giant cells were derived from nerve cells but not from ependymal cells. In hamsters studied by Waksman et al. (7), however, giant cells were clearly derived from either ependymal or nerve cells. Specific measles antigens were demonstrated by the fluorescent antibody staining predominantly structures in nerve cells, only rarely in glia cells, but not in other types of cell in the brain. Specific fluorescence was always demonstrated in the cytoplasm and not in the nucleus, as in infected F L cells. This is an example of virus variation and its further studies would shed light on the genetics of measles virus. On the other hand, it would give a virus-host system of great value for the study on interaction of measles virus with the central nervous system, particularly in connection with the problem of post-infectious measles encephalomyelitis. The mouse adapted virus might be utilized also for the evaluation of biologic or therapeutic agents affecting measles. A r c h l y f. V i r u s f o r s c h u n g , Bd. X I V , I t . 5
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Matumoto et al.: A Neurotropic Variant of Measles Virus Summary
Strain Sugiyama, adapted to F L cells after 6 passages in primary monkey renal ceils, acquired the ability to propagate serially in the brain of suckling mice somewhere between the 49th and 76th FL passage. The virus produced a lethal spastic paralysis in about half of mice at the early passages and in almost 100% from the 9th passage on. Mice were nearly 100% susceptible within 5 days after birth and almost 100% resistant at the age of 7 days or over. Extraneural routes of inoculation were ineffective for infection. Histologic changes were found only in the cerebrum, not in the cerebellum or spinal cord, nor in other visceral organs, and consisted of degeneration of nerve cells with or without formation of inclusions characteristic of measles, multinuclear giant cell formation by fusion of nerve cells, and proliferation of glia cells. Fluorescent antibody study revealed remarkable specificity of nerve cell involvement. Specific measles antigens were demonstrated predominantly in nerve cells, only rarely in glia cells, but not in other types of cell. Acknowledgements : The authors are greatly indebted to Dr. A. Kawamura and Dr. H. Nagahama for preparing fluorescent antibody.
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References CarlstrSm, G.: Comparative studies on measles and distemper viruses in suckling mice. Arch. ges. Virusforsch. 8~ 527--538 (1958). Enders, J. I~., and T. C. Peebles: Propagation in tissue cultures of cytopathogenic agents from patients with measles. Proc. Soe. Exp. Biol. Med. 86, 277--286 (1954). Imagawa, D. T., and J. M. Adams: Propagation of measles virus in suckling mice. Proc. Soc. Exp. Biol. ~[cd. 98, 567--569 (1958). Kgrber, G.: Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. Arch. Exp. Path. Pharm. 162, 480--498 (1931). 2l/latumoto, M., M. Mutai, H. Ogiwara, I. Nishi, 2V. Kusano, et Y. Aoyama: Isolement du virus do la rougeolc en culture du tissu r~nal du singe. C. R. Soc. Biol. 158, 879--883 (1959). Matumoto, M., M. Mutai, et H. Ogiwara: Prolif6ration du virus rongeoleux cn culture de cellules rdnales bovines. C.R. Soe. Biol. 155~ 1192--1195 (1961). Waksman, B. H., T. Burnstein, and R. D. Adams: Histologic study of the encephalomyelitis produced in hamsters by a neurotropic strain of measles. J. Neurol. Exp. Path. 21~ 25--49 (1962).