Clin. exp. Immunol. (1992) 89, 414-418
Cells with natural killer activity in human rabies T. PANPANICH, T. HEMACHUDHA, S. PIYASIRISILP, S. MANATSATHIT*, H. WILDE & P. PHANUPHAK Queen Saovabha Memorial Institute, Thai Red Cross Society and Faculty of Medicine, Chulalongkorn University Hospital, Bangkok, and *Bumrasanaradura Hospitalfor Infectious Diseases, Nonthaburi, Thailand
(Acceptedfor publication 3 June 1992)
SUMMARY Cytotoxic lymphocyte function in 13 patients with rabies was studied by counting the number of CD56 cells and assessing natural killer (NK) cell activity. There was no significant difference in the number of killer cells between rabies patients and 31 normal controls (P > 0 05). Two of six non-fatal encephalitic patients due to causes other than rabies had reduced CD56 numbers. Base-line NK cell responses versus K562 cell targets did not differ among the normal control and rabies groups (P> 0-05). Study of the non-rabies encephalitis group showed heterogeneous results with wide variation. Significant enhancement of NK activity was seen in four rabies patients and in 10 normal control subjects tested after interferon-alpha (IFN-ot) and IL-2. None of the four patients with encephalitis due to causes other than rabies showed such enhancement. Our results suggest that NK cells of rabies patients are not fully stimulated and that this might contribute to the virulence of rabies. The cause of this phenomenon remains unknown. Keywords human rabies natural killer cells interferon IL-2 encephalitis Survival in man after onset of clinical symptoms of rabies usually ranges from a few days to 1 week. However, in individuals who have a paralytic presentation, survival periods of more than 2 weeks may be observed [10]. Under the circumstance of a rapidly progressive disease course, NK cells might be able to retard dissemination of virus. In this study the number of circulating CD56 cells and NK cell function were compared in rabies and surviving non-rabies encephalitic patients. The purpose of the study was to determine whether the response of NK cells was intact in rabies patients.
INTRODUCTION Factors that determine the virulence of rabies are poorly understood. We have found that defects in recognition of rabies nucleocapsid protein may be responsible for a poor rabies neutralizing antibody response [1]. Rabies patients who present with paralysis rather than with encephalitis have a diminished cell-mediated immune response. This is evident from decreased serum levels of soluble IL-2 receptor, IL-6 (unpublished data), and absence of lymphocyte proliferation to rabies antigen [2]. Wiktor et al. [3-5] have previously shown that infection of mice with an attenuated strain of virus induced a rabies-specific cytotoxic T lymphocyte response, whereas infection with a virulent strain did not. The number of circulating CD57 cells was markedly diminished in rabies patients [6]. Recent evidence in animals has identified a potentially important role for natural killer (NK) cells in host defences against viral infections other than rabies [7,8]. This is supported by the discovery of an individual with recurrent herpes infection whose only demonstrable immunological defect was the absence of NK cells [9]. NK cells can lyse virus-infected cells without any apparent previous sensitization and without involvement of
PATIENTS AND METHODS Patients Thirteen patients with rabies were admitted to Bumrasanaradura Hospital between November 1989 and November 1990. made if there was a reliable history of a dog or cat bite, typical tyia mad iferwasa ciia aiettosicuigarpoihdohbaad or inspiratory spasms and a fatal outcome [10]. Six individuals with CNS infections other than rabies (herpes simplex encephalitis (n = 2), tuberculous meningoencephalitis (n =2) and unidentified viral etiologies (n = 2) and 31 healthy adults served as controls. All patients in the non-rabies group were admitted to Chulalongkorn University Hospital during the same period. The diagnoses were made by typical cerebrospinal fluid profiles, radiologic imaging and identification or detection of antibodies to the responsible organisms and/or improvement after specific treatment.
nreliabl hsrypofiad ordcathbit
major histocompatibility antigens. They respond rapidly to virus challenge and mount proliferative and cytolytic responses .v . I, Corspnene Poeso TiavtHeahuh, uenSo vabha Memorial Institute, WHO Collaborating Centre for Research on Rabies Pathogenesis and Prevention, Thai Red Cross Society, Rama IV Road, Bangkok 10330, Thailand.
414
415
Cells with NK activity in human rabies NK assays Blood was collected, heparinized (20 U/ml) and the peripheral blood mononuclear cells (PBMC) were separated on FicollHypaque (Pharmacia Fine Chemicals, Piscataway, NJ) gradients. Cells were washed and resuspended in RPMI 1640 (GIBCO, Grand Island, NY) supplemented with 5% fetal bovine serum (FBS; GIBCO), 10 mm HEPES, pH 7 4, and gentamicin (50 pg/ml). K562 cells (gift of D. E. Griffin, The Johns Hopkins University School of Medicine, MD) were used as target cells [11] and were grown in RPMI/5% FBS, passed the day before use and labelled with 51Cr (Amersham, Amersham, UK). The assay was conducted in round-bottomed microtitre plates as previously described [12] using 5 x 103 target cells/well and a PBMC effector-to-target cell ratio (E: T) of 100: 1, 30: 1, 10: 1, 3: 1 and 1: 1. PBMC from controls and patients with encephalitis were assayed simultaneously. Assays were performed in triplicate in a volume of 200 id/well. Chromium release was measured after 4 h at 37°C. Specific lysis was calculated using 1% SDS to determine the maximum lysis. NK cell activity was expressed as lytic units (LU)/107 PBMC as determined by least squares analysis derived from per cent specific lysis of all E: T ratios. One LU was defined as the number of effector cells required for 20% specific lysis of 5 x 103 target cells. In vitro activation of cytotoxic cells PBMC in RPMI/5% FBS were cultured overnight at a concentration of 106 cells/ml in the presence of 100 U/ml of recombinant IL-2 (Genzyme, MA) and interferon-alpha (IFN-a; Schering, NJ) and then assayed for lytic activity against 5'Cr-labelled K562 cells as described above.
Immunoperoxidase staining PBMC obtained on the first day of admission were isolated by Ficoll-Hypaque density gradient centrifugation and then sedimented on to slides by cytocentrifugation (Shandon Southern Instruments, Sewickley, PA). Cells were stained by the avidinbiotin complex method [6], using mouse MoAb, Leu- 19 (CD56) (Becton, MD). Briefly, cells were incubated for 20 min with normal horse serum to block non-specific staining, washed in PBS, and incubated for 45 min with Leu-19 at a dilution of 1: 100 in PBS. Normal mouse serum at 1: 1000 dilution served as
a control. After two 5-min rinses in PBS, cells were incubated for 30 min in biotinylated horse anti-mouse IgG serum diluted 1:100 (Vector, CA). After further washing, slides were incu-
bated for 30 min in 1% hydrogen peroxide in methanol, then washed and incubated for 30 min in a freshly prepared complex of avidin biotinylated horseradish peroxidase (Dakopatts, Denmark). Slides were washed again and incubated for 20 min with substrate of diaminobenzidine (0-5 mg/ml and hydrogen peroxide 0 1% in PBS). After rinsing, the stain was darkened by soaking in 0 5% CuSO4 in 0-15 M NaCl for 5 min, rinsed again and counterstained with Gill's haematoxylin. Cells were examined under oil immersion ( x 100) and quantified as the percentage of 200 PBMC.
Statistical analysis Data were analysed using the non-parametric Mann-Whitney U-test for NK number and function and the Wilcoxon signedrank test for in vitro activation.
RESULTS Clinical data Ten rabies patients had the classical encephalitic manifestation with alternating intervals of confusion and lucid calm, aerophobia, hydrophobia, and evidence of autonomic disturbances. The remaining three cases had paralysis. All three had pure motor weakness of all limbs as an early presentation. Phobic spasms were absent but inspiratory spasms were observed during the terminal stage. All patients in both groups were alert and rational at the time blood samples were collected, usually on admission. The intervals between onset of disease and day at which blood samples were taken were roughly similar in all patients (an average of 3 days in encephalitic and 4 days in the paralytic group). The intervals between blood collection and death were 1-3 days (Table 1). Lumbar punctures were performed in five patients. They revealed only mild pleocytosis of not more than 1O cells/mm3, thus eliminating an opportunity to study NK response in the CSF compartment. Six patients with encephalitis other than rabies also had their blood collected at the time of admission, 2-3 days after onset of the disease.
Table 1. Characteristics of patients with rabies
Patient no.
1 2 3 4 5 6 7 8 9
10 11 12 13
Sex
Incubation period (months)
Days from onset to admission
Days from admission to death
6 33 28 51 18 31 58 65 23
Female Male Male Female Male Female Female Male Male
0-3 2 4 2 2 1 5 3 2
3 4 2 3 4 3 6 5 1
1 2 2 3 1 2 1 2 3
11
Male
8
7 63 52
Female Male Female
0-5
41 41
2 3
4 2
Clinical type
Age (years)
Encephalitis Encephalitis Encephalitis Encephalitis Paralysis Encephalitis Paralysis Encephalitis Encephalitis Paralysis Encephalitis Encephalitis Encephalitis
2 2
416
T. Panpanich et al. 110
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(n )13) (n(31) Fig. 1. Percentage of circulating CD56 cells in individuals with rabies
1 -
20
-
and controls. There was no significant difference among these two groups (P> 005). Means + sem. are indicated for each group. I0
These patients
although
usually obtunded. However, all survived 0 neurological residua. the
were
some had
(n: 3i
NK number
Thengrebwas noasignfcnts among values~~ ~~ (l4K3
diffrenc
rabies patients
Rabnes
1
indth cnumbrofs CD456 ~ ~ ~ ~ ~
cells
± 24 s~d.) and controls (145 ± 18) -
~
~
(P> 0a05) (Fig. 1). In the non-rabies encephalitic group,
numbers varied between 8% and 16%. Two patients had a diminished number of CD56 cells (below 2 s.d. from the mean of values from 31 controls). NK activity NK activity expressed in LU/ 107 PBMC for rabies patients was compared with the normal control group. Rabies patients as a group had NK activity comparable to that of normal controls (45-3 + 18-8 versus 55-4+22 3) (P> 0 05) (Fig. 2). NK activity ranged between 16 and 108 LU/10 PBMC in the non-rabies group. There was no correlation between number of CD56 cells and NK activity in this group, as demonstrated in Fig. 3. Two patients with tuberculous meningoencephalitis who had a normal CD56 number appeared to have significantly diminished NK activity. On the other hand, two patients with herpes simplex and viral encephalitis of unidentified cause, whose NK activities were within normal range, had significantly diminished CD56 numbers (Fig. 3). In vitro activation for cytotoxic cells In vitro cultures of PBMC from four rabies, four non-rabies encephalitic patients and from 10 normal controls with recombinant IL-2 and IFN-x were performed. Lytic activity in all rabies patients increased after stimulation with IL-2 (P < 005) and IFN-a (P < 0-01). Similar findings were found in normal controls with IL-2 (P< 001) and IFN-cx (P 4005). Means±s.e.m. are indicated for each group.
enhancement of NK cell activity was not evident in non-rabies encephalitic patients which already appeared to be maximally stimulated (Fig. 4). DISCUSSION
Neutralizing antibody has been held responsible for abortive infection in mice that are genetically resistant to rabies virus infection [13,14]. This, however, does not exclude the importance of the killer cell system. No correlation was found between appearance of serum antibody and survival in mice [3-5], although such correlation was demonstrated in vaccine efficacy trials in dogs [15]. Rabies nucleocapsid protein is essential for the elicitation of neutralizing antibody and protects animals from rabies virus challenge [16-18]. Some of these animals can survive without developing neutralizing antibody, thus demonstrating the importance of cellular immunity in rabies. Lack of cytotoxic killing function, using spleen cells as effectors, parallels mortality in mice [3-5]. Cells with killing function can be classified into fast-responding NK, cytotoxic T and K cells. NK cells require none of the MHC restriction nor previous contact or sensitization with the antigen. Cytotoxic T cells bind targets while recognizing antigen and MHC determinant. K
417
Cells with NK activity in human rabies
of antigen processing or it may involve interaction between macrophages and T cells, clonal proliferation and differentia120
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Cells with natural killer activity in human rabies T. PANPANICH, T. HEMACHUDHA, S. PIYASIRISILP, S. MANATSATHIT*, H. WILDE & P. PHANUPHAK Queen Saovabha Memorial Institute, Thai Red Cross Society and Faculty of Medicine, Chulalongkorn University Hospital, Bangkok, and *Bumrasanaradura Hospitalfor Infectious Diseases, Nonthaburi, Thailand
(Acceptedfor publication 3 June 1992)
SUMMARY Cytotoxic lymphocyte function in 13 patients with rabies was studied by counting the number of CD56 cells and assessing natural killer (NK) cell activity. There was no significant difference in the number of killer cells between rabies patients and 31 normal controls (P > 0 05). Two of six non-fatal encephalitic patients due to causes other than rabies had reduced CD56 numbers. Base-line NK cell responses versus K562 cell targets did not differ among the normal control and rabies groups (P> 0-05). Study of the non-rabies encephalitis group showed heterogeneous results with wide variation. Significant enhancement of NK activity was seen in four rabies patients and in 10 normal control subjects tested after interferon-alpha (IFN-ot) and IL-2. None of the four patients with encephalitis due to causes other than rabies showed such enhancement. Our results suggest that NK cells of rabies patients are not fully stimulated and that this might contribute to the virulence of rabies. The cause of this phenomenon remains unknown. Keywords human rabies natural killer cells interferon IL-2 encephalitis Survival in man after onset of clinical symptoms of rabies usually ranges from a few days to 1 week. However, in individuals who have a paralytic presentation, survival periods of more than 2 weeks may be observed [10]. Under the circumstance of a rapidly progressive disease course, NK cells might be able to retard dissemination of virus. In this study the number of circulating CD56 cells and NK cell function were compared in rabies and surviving non-rabies encephalitic patients. The purpose of the study was to determine whether the response of NK cells was intact in rabies patients.
INTRODUCTION Factors that determine the virulence of rabies are poorly understood. We have found that defects in recognition of rabies nucleocapsid protein may be responsible for a poor rabies neutralizing antibody response [1]. Rabies patients who present with paralysis rather than with encephalitis have a diminished cell-mediated immune response. This is evident from decreased serum levels of soluble IL-2 receptor, IL-6 (unpublished data), and absence of lymphocyte proliferation to rabies antigen [2]. Wiktor et al. [3-5] have previously shown that infection of mice with an attenuated strain of virus induced a rabies-specific cytotoxic T lymphocyte response, whereas infection with a virulent strain did not. The number of circulating CD57 cells was markedly diminished in rabies patients [6]. Recent evidence in animals has identified a potentially important role for natural killer (NK) cells in host defences against viral infections other than rabies [7,8]. This is supported by the discovery of an individual with recurrent herpes infection whose only demonstrable immunological defect was the absence of NK cells [9]. NK cells can lyse virus-infected cells without any apparent previous sensitization and without involvement of
PATIENTS AND METHODS Patients Thirteen patients with rabies were admitted to Bumrasanaradura Hospital between November 1989 and November 1990. made if there was a reliable history of a dog or cat bite, typical tyia mad iferwasa ciia aiettosicuigarpoihdohbaad or inspiratory spasms and a fatal outcome [10]. Six individuals with CNS infections other than rabies (herpes simplex encephalitis (n = 2), tuberculous meningoencephalitis (n =2) and unidentified viral etiologies (n = 2) and 31 healthy adults served as controls. All patients in the non-rabies group were admitted to Chulalongkorn University Hospital during the same period. The diagnoses were made by typical cerebrospinal fluid profiles, radiologic imaging and identification or detection of antibodies to the responsible organisms and/or improvement after specific treatment.
nreliabl hsrypofiad ordcathbit
major histocompatibility antigens. They respond rapidly to virus challenge and mount proliferative and cytolytic responses .v . I, Corspnene Poeso TiavtHeahuh, uenSo vabha Memorial Institute, WHO Collaborating Centre for Research on Rabies Pathogenesis and Prevention, Thai Red Cross Society, Rama IV Road, Bangkok 10330, Thailand.
414
415
Cells with NK activity in human rabies NK assays Blood was collected, heparinized (20 U/ml) and the peripheral blood mononuclear cells (PBMC) were separated on FicollHypaque (Pharmacia Fine Chemicals, Piscataway, NJ) gradients. Cells were washed and resuspended in RPMI 1640 (GIBCO, Grand Island, NY) supplemented with 5% fetal bovine serum (FBS; GIBCO), 10 mm HEPES, pH 7 4, and gentamicin (50 pg/ml). K562 cells (gift of D. E. Griffin, The Johns Hopkins University School of Medicine, MD) were used as target cells [11] and were grown in RPMI/5% FBS, passed the day before use and labelled with 51Cr (Amersham, Amersham, UK). The assay was conducted in round-bottomed microtitre plates as previously described [12] using 5 x 103 target cells/well and a PBMC effector-to-target cell ratio (E: T) of 100: 1, 30: 1, 10: 1, 3: 1 and 1: 1. PBMC from controls and patients with encephalitis were assayed simultaneously. Assays were performed in triplicate in a volume of 200 id/well. Chromium release was measured after 4 h at 37°C. Specific lysis was calculated using 1% SDS to determine the maximum lysis. NK cell activity was expressed as lytic units (LU)/107 PBMC as determined by least squares analysis derived from per cent specific lysis of all E: T ratios. One LU was defined as the number of effector cells required for 20% specific lysis of 5 x 103 target cells. In vitro activation of cytotoxic cells PBMC in RPMI/5% FBS were cultured overnight at a concentration of 106 cells/ml in the presence of 100 U/ml of recombinant IL-2 (Genzyme, MA) and interferon-alpha (IFN-a; Schering, NJ) and then assayed for lytic activity against 5'Cr-labelled K562 cells as described above.
Immunoperoxidase staining PBMC obtained on the first day of admission were isolated by Ficoll-Hypaque density gradient centrifugation and then sedimented on to slides by cytocentrifugation (Shandon Southern Instruments, Sewickley, PA). Cells were stained by the avidinbiotin complex method [6], using mouse MoAb, Leu- 19 (CD56) (Becton, MD). Briefly, cells were incubated for 20 min with normal horse serum to block non-specific staining, washed in PBS, and incubated for 45 min with Leu-19 at a dilution of 1: 100 in PBS. Normal mouse serum at 1: 1000 dilution served as
a control. After two 5-min rinses in PBS, cells were incubated for 30 min in biotinylated horse anti-mouse IgG serum diluted 1:100 (Vector, CA). After further washing, slides were incu-
bated for 30 min in 1% hydrogen peroxide in methanol, then washed and incubated for 30 min in a freshly prepared complex of avidin biotinylated horseradish peroxidase (Dakopatts, Denmark). Slides were washed again and incubated for 20 min with substrate of diaminobenzidine (0-5 mg/ml and hydrogen peroxide 0 1% in PBS). After rinsing, the stain was darkened by soaking in 0 5% CuSO4 in 0-15 M NaCl for 5 min, rinsed again and counterstained with Gill's haematoxylin. Cells were examined under oil immersion ( x 100) and quantified as the percentage of 200 PBMC.
Statistical analysis Data were analysed using the non-parametric Mann-Whitney U-test for NK number and function and the Wilcoxon signedrank test for in vitro activation.
RESULTS Clinical data Ten rabies patients had the classical encephalitic manifestation with alternating intervals of confusion and lucid calm, aerophobia, hydrophobia, and evidence of autonomic disturbances. The remaining three cases had paralysis. All three had pure motor weakness of all limbs as an early presentation. Phobic spasms were absent but inspiratory spasms were observed during the terminal stage. All patients in both groups were alert and rational at the time blood samples were collected, usually on admission. The intervals between onset of disease and day at which blood samples were taken were roughly similar in all patients (an average of 3 days in encephalitic and 4 days in the paralytic group). The intervals between blood collection and death were 1-3 days (Table 1). Lumbar punctures were performed in five patients. They revealed only mild pleocytosis of not more than 1O cells/mm3, thus eliminating an opportunity to study NK response in the CSF compartment. Six patients with encephalitis other than rabies also had their blood collected at the time of admission, 2-3 days after onset of the disease.
Table 1. Characteristics of patients with rabies
Patient no.
1 2 3 4 5 6 7 8 9
10 11 12 13
Sex
Incubation period (months)
Days from onset to admission
Days from admission to death
6 33 28 51 18 31 58 65 23
Female Male Male Female Male Female Female Male Male
0-3 2 4 2 2 1 5 3 2
3 4 2 3 4 3 6 5 1
1 2 2 3 1 2 1 2 3
11
Male
8
7 63 52
Female Male Female
0-5
41 41
2 3
4 2
Clinical type
Age (years)
Encephalitis Encephalitis Encephalitis Encephalitis Paralysis Encephalitis Paralysis Encephalitis Encephalitis Paralysis Encephalitis Encephalitis Encephalitis
2 2
416
T. Panpanich et al. 110
100 2 30 QC~~~~~~~~~~~~~~~~~~~~ -
90
0
Co
0
80
0
~~~~~~~~~~~~~~~~~~~~~~~~
C,
+
0
~~~~~~~~~~~~~~~~~~0
(D c
20 _
70 _
10
0
C:
0~~~~~~~
00
0* 000 *000 00
X 40 60..
00
No0ma
_ -
.0 A
R 0
00
0
30
Rabies
Normal
40 10
(n )13) (n(31) Fig. 1. Percentage of circulating CD56 cells in individuals with rabies
1 -
20
-
and controls. There was no significant difference among these two groups (P> 005). Means + sem. are indicated for each group. I0
These patients
although
usually obtunded. However, all survived 0 neurological residua. the
were
some had
(n: 3i
NK number
Thengrebwas noasignfcnts among values~~ ~~ (l4K3
diffrenc
rabies patients
Rabnes
1
indth cnumbrofs CD456 ~ ~ ~ ~ ~
cells
± 24 s~d.) and controls (145 ± 18) -
~
~
(P> 0a05) (Fig. 1). In the non-rabies encephalitic group,
numbers varied between 8% and 16%. Two patients had a diminished number of CD56 cells (below 2 s.d. from the mean of values from 31 controls). NK activity NK activity expressed in LU/ 107 PBMC for rabies patients was compared with the normal control group. Rabies patients as a group had NK activity comparable to that of normal controls (45-3 + 18-8 versus 55-4+22 3) (P> 0 05) (Fig. 2). NK activity ranged between 16 and 108 LU/10 PBMC in the non-rabies group. There was no correlation between number of CD56 cells and NK activity in this group, as demonstrated in Fig. 3. Two patients with tuberculous meningoencephalitis who had a normal CD56 number appeared to have significantly diminished NK activity. On the other hand, two patients with herpes simplex and viral encephalitis of unidentified cause, whose NK activities were within normal range, had significantly diminished CD56 numbers (Fig. 3). In vitro activation for cytotoxic cells In vitro cultures of PBMC from four rabies, four non-rabies encephalitic patients and from 10 normal controls with recombinant IL-2 and IFN-x were performed. Lytic activity in all rabies patients increased after stimulation with IL-2 (P < 005) and IFN-a (P < 0-01). Similar findings were found in normal controls with IL-2 (P< 001) and IFN-cx (P 4005). Means±s.e.m. are indicated for each group.
enhancement of NK cell activity was not evident in non-rabies encephalitic patients which already appeared to be maximally stimulated (Fig. 4). DISCUSSION
Neutralizing antibody has been held responsible for abortive infection in mice that are genetically resistant to rabies virus infection [13,14]. This, however, does not exclude the importance of the killer cell system. No correlation was found between appearance of serum antibody and survival in mice [3-5], although such correlation was demonstrated in vaccine efficacy trials in dogs [15]. Rabies nucleocapsid protein is essential for the elicitation of neutralizing antibody and protects animals from rabies virus challenge [16-18]. Some of these animals can survive without developing neutralizing antibody, thus demonstrating the importance of cellular immunity in rabies. Lack of cytotoxic killing function, using spleen cells as effectors, parallels mortality in mice [3-5]. Cells with killing function can be classified into fast-responding NK, cytotoxic T and K cells. NK cells require none of the MHC restriction nor previous contact or sensitization with the antigen. Cytotoxic T cells bind targets while recognizing antigen and MHC determinant. K
417
Cells with NK activity in human rabies
of antigen processing or it may involve interaction between macrophages and T cells, clonal proliferation and differentia120
20
TS
90
5
_i g a.
v
HS o
00
r
\
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\
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,
*
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