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 ~  Abstract
 ~  Materials and Me...
 ~  Results
 ~  Discussion
 ~  Aknowledgements
 ~  References

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SPECIAL ARTICLE
Year : 2001  |  Volume : 19  |  Issue : 3  |  Page : 127-131
 

Neuronal apoptosis in herpes simplex virus - 1 Encephalitis (HSE)


Jhaveri Microbiology Centre, L. V. Prasad Eye Institute, Nizam's Institute of Medical Sciences, Hyderabad, India

Correspondence Address:
Jhaveri Microbiology Centre, L. V. Prasad Eye Institute, Nizam's Institute of Medical Sciences, Hyderabad, India

 ~ Abstract 

Herpes simplex virus infections are encountered often due to their ubiquitous nature. Common sites involved include skin, mucous membrane, genitalia, eye and the nervous system. HSV infection of the central nervous system can be life threatening. Little is known about the pathogenesis of this cataclysmic disease, at the cellular level. Virus induced apoptosis may play a role in the molecular pathogenesis of encephalitis. This study aims to detect the presence of apoptosis: a) In the brain tissue obtained at autopsy from a patient who succumbed to Herpes simplex virus - 1 encephalitis (HSE) and b) In a human glioblastoma cell line (SNB 19). Wedge tissue samples were obtained from the inferior surface of the frontal lobe and fixed in buffered formalin. Tissue sections were stained with haematoxylin and eosin for histopathological analysis. An indirect immunoperoxidase assay was performed for the detection of HSV -1 antigen in the tissue sections. Apoptosis in the brain tissue was detected employing the TUNEL assay (Terminal deoxynucleotidyl Transferase (TdT) mediated deoxy Uridine Triphosphate Nick End Labeling) using a commerically available kit (TdT Fragel DNA fragmentation detection kit, Oncogene Research Products, CA). HSV-1 induced apoptosis of SNB 19 cells were detected in-vitro by: a) Membrane blebbing assay and b) Hoechst 33258 staining. Classical features of viral encephalitis including the presence of intranuclear inclusions, neuronal loss and perivascular cuffing were seen in the tissue sections. The immunoperoxidase assay revealed the presence of abundant viral antigen in the neurons, microglial and satellite cells. TUNEL assay revealed many apoptotic neurons, microglial and satellite cells. In-vitro assays showed evidence of HSV-1 induced apoptosis in the SNB 19 cell line. These results suggest that virus induced apoptosis may play a role in the molecular pathogenesis of HSE. Further studies are warranted to elucidate the role of HSV-1 induced apoptosis, especially employing cell lines of neuronal origin.

How to cite this article:
Athmanathan S, Vydehi B V, Sundaram C, Vemuganti G K, Murthy J M. Neuronal apoptosis in herpes simplex virus - 1 Encephalitis (HSE). Indian J Med Microbiol 2001;19:127-31


How to cite this URL:
Athmanathan S, Vydehi B V, Sundaram C, Vemuganti G K, Murthy J M. Neuronal apoptosis in herpes simplex virus - 1 Encephalitis (HSE). Indian J Med Microbiol [serial online] 2001 [cited 2019 Aug 23];19:127-31. Available from: http://www.ijmm.org/text.asp?2001/19/3/127/8145


Herpes simplex virus-1 (HSV-1) and Herpes simplex virus-2 (HSV-2) infections are virtually universal in man, since these viruses are ubiquitous.[1] Infections often involve the eye, mucous membrane, brain, skin, genitalia and recurrences are quite common. HSV infection of the central nervous system can be life threatening. Five categories of disease of the CNS have been ascribed to HSV.[1] They include: a) Chronic nervous or psychiatric illnesses, b) Meningitis, c) Mild diffuse encephalitis, d) Severe diffuse encephalitis, e) Focal (necrotising) encephalitis. Herpes simplex encephalitis (HSE) could be a devastating CNS infection, especially when it occurs as severe diffuse encephalitis or as necrotising encephalitis. Little is known about the pathogenesis of this cataclysmic disease, at the cellular level. Virus induced apoptosis may play a role in the molecular pathogenesis of encephalitis. Recent studies have shown that virus induced apoptosis is a mechanism of cell death in viral infections of the CNS.[2],[3],[4] These studies have attributed the ensuing clinical manifestations to this phenomenon.
HSV-1 induced apoptosis of neural cells has not been extensively studied. The aim of this study was to detect HSV-1 induced apoptosis: a) In the brain tissue obtained at autopsy from a case of HSE and b) In a human glioblastoma cell culture model.

 ~ Materials and Methods Top

Brain tissues
Brain tissues were obtained at autopsy from a 40 year old deceased male who was diagnosed as a case of HSE based on clinical history and CT findings. Wedge tissues were obtained from the inferior surface of the frontal lobe and fixed in buffered formalin.
Histopathology
Tissue sections were obtained from the formalin fixed, paraffin embedded brain tissues and were stained with haematoxylin and eosin for histopathological analysis.
HSV-1 antigen detection by immunohistochemistry
Viral antigen was detected in the tissue sections employing an indirect immunoperoxidase technique. Briefly, sections were hydrated and blocked with 3% skimmed milk powder in PBS with 0.5% tween 20. Endogenous peroxidase was quenched using 3%H2O2. Sections were incubated with polyclonal anti-HSV-1 antibody (Dako, Carpinteria, LA) followed by biotinylated anti-rabbit immunoglobulins as the secondary antibody. Sections were then incubated with avidin peroxidase followed by H2O2 and di-amino benzidine (DAB) as the substrate-chromogen complex. Sections were counterstained with haematoxylin, dehydrated, mounted and observed by light microscopy.
Detection of Apoptosis
Apoptosis in the brain tissue was detected employing the TUNEL assay[5] (Terminal deoxynucleotidyl Transferase (TdT) mediated deoxy Uridine Triphosphate Nick End Labeling) using a commerically available kit (TdT Fragel DNA fragmentation detection kit, Oncogene Research Products, CA) as per manufacturer's instruction. Briefly, sections were obtained from formalin fixed brain tissue, deparaffinized and hydrated using graded alcohols. The sections were then permeabilised with proteinase K (2 mg/ml, 1:100 in 10 mM Tris, pH 8) solution for 20 minutes at room temperature. Endogenous peroxidase was quenched with 30% hydrogen peroxide (1:10 in methanol). Sections were incubated with the labeling reaction mixture consisting of TdT and biotinylated nucleotides for 75 minutes at 37C and the reaction was terminated with the stop buffer. Labeled DNA fragments were visualized by adding streptavidin-horseradish peroxidase conjugate, incubating for 30 minutes and developing with 3, 3-diaminobenzidine tetrahydrochloride (DAB). Sections were counterstained with 0.3 % methyl green solution. TUNEL positive cells (containing labeled DNA fragments) showed dark brown staining of the nucleus suggesting the internucleosomal cleavage of DNA. All other cells appeared green due to the counterstain. Tissues other than brain (donor cornea) were processed similarly and were used as controls.
In-vitro assays for the detection HSV-1 induced apoptosis using a glioblastoma cell culture model.
Detection of apoptosis in a cell culture model was done employing two different methods using a human glioblastoma cell line (SNB 19)(Kind gift from Dr. Sriram Rajagopal, Dr. Reddy's Research Foundation, Hyderabad, A.P.).
A. Membrane blebbing
SNB 19 cells were infected with HSV-1 (ATCC, V-539) at a MOI of 1 and cells were observed for membrane blebbing6 at 48 hours using an inverted microscope (TO41, Olympus, Japan) by phase contrast microscopy. Uninfected cells were used as controls.
B. Analysis of nuclear morphology
HSV-1 infected SNB 19 cells were harvested at 48 hours post infection and stained with Hoechst 33258 (bis-Benzimide) as described previously,[7] with minor modifications. Briefly, a stock solution (10 g/mL) of bis-Benzimide (Sigma, St.Louis, MO) was prepared using milli-Q water. Both infected and uninfected cells (floating and attached cells) collected at 48 hours following trypsinization, were washed with phosphate buffered saline, pH 7.2 and re-suspended in 100 ml of 70% ethyl alcohol for fixation. Cell suspension (3 L) was mixed with 2 L of the stock solution of bis-Benzimide on a glass slide. A coverslip was placed over the suspension and cells were incubated for 10 minutes at room temperature. The preparation was observed under a fluorescence microscope (BH 2-RFC, Olympus, Japan) at a wave length of 365 nm. A minimum of 500 cells were counted in three such preparations to calculate the percentage of cells showing apoptotic changes. Apototic changes were detected by the presence of chromatin condensation and apoptotic bodies. Uninfected cells were processed similarly and used as controls.

 ~ Results Top

Gross pathology
Gross pathological examination of the brain slices revealed haemorrhagic infarcts at the level of internal capsule
(Fig. A), hippocampus and brain stem.
Histopathology
Haematoxylin and eosin stained sections revealed neuronal loss (Fig. B), areas where neurons were preserved
(Fig. C), intranuclear inclusion (Fig. D) and perivascular cuffing (Fig. E), all suggestive of viral encephalitis.
Viral antigen detection
HSV-1 antigen was detected in the neurons, microglial cells and satellite cells. The presence of viral antigen showed a patchy distribution (Fig. F). Many cortical neurons were intensely stained (Fig. G).
Detection of apoptosis
TUNEL positive cells (Figs. H and J) were detected among the cortical neurons, which also showed a patchy distribution. There were regions where the neurons were [Figure:1]
A. Haemorrhagic infarcts at the level of internal capsule.
B. Section from the left frontal lobe showing an area of neuronal loss, H&E, X 500.
C. Section from the left frontal lobe showing an area of preserved neurons, H& E, X 500.
D. Section from the left frontal lobe showing a neuron with intranuclear eosinophilic inclusion (arrow), H & E, X 1250.
E. Section from the left frontal lobe showing perivascular cuffing, H & E, X 500.
F. Immunohistochemical localization of HSV-1 antigen. Note the patchy distribution of the viral antigen (arrow). Immunohistochemistry, X 50.
G. Immunohistochemical localization of HSV-1 antigen. Note the presence of intense staining of neurons (arrows). Immunohistochemistry, X 500.
H. Brain tissue stained by the TUNEL assay. Note the presence of apoptotic cells (Arrows, nuclei are stained brown). X 500.
I. Brain tissue stained by the TUNEL assay. Note the absence of apoptotic cells, X 500.
J. Brain tissue stained by the TUNEL assay. A higher magnification view of an apoptotic neuron (arrow). Note the intense staining of the nucleus. X 1250.
K. HSV- 1 infected SNB 19 cells. Note the presence of membrane blebbing (arrows), loss of monolayer and rounding of cells (CPE). Phase contrast microscopy, X 400.
L. Monolayer of SNB 19 cells. Phase contrast microscopy, X 100.
negative for TUNEL staining [Figure:1]. In addition, many glial and satellite cells showed apoptotic changes (not shown).
In-vitro assays for the detection HSV-1 induced apoptosis using a glioblastoma cell culture model.
A. Membrane blebbing
More than 80% of the HSV-1 infected cells showed cytopathic effect at the end of 48 hours post-infection and more than 50% of these cells showed membrane blebbing, a characteristic feature of cells undergoing apoptosis.[6] These features were not seen in uninfected cells.
B. Analysis of nuclear morphology
Analysis of nuclear morphology following Hoechst 33258 staining of HSV-1 infected cells showed the presence of nuclear condensation and apoptotic bodies, a characteristic nuclear feature of cells undergoing apoptosis,[7] in more than 50% of the cells. Uninfected cells did not show such changes.

 ~ Discussion Top

This report provides evidence for the presence of apoptosis in HSE. Apoptosis or programmed cell death is a fundamental process that occurs during development, homeostasis, and wound healing in the tissues of essentially all multicellular organisms.[8],[9] Apoptosis has been incriminated in many diseases including infections, cancer, autoimmune and neurodegenerative disorders.
All cells of higher organisms express the molecular machinery necessary to undergo apoptosis. Some of the classical stimulators of apoptosis are viral infection, growth factor deprivation, extensive DNA damage, ionising radiation, corticosteroids and specific apoptosis associated cytokines.[10]
Viruses from several different families are able to exploit their host's cell death programmes to maximize virus survival. Considering the evolution of such strategies, it has been suggested that the virus should inhibit apoptosis, in order to prolong the survival of the cells resulting in a highly productive infection in terms of progeny virions. On the other hand, the host should stimulate apoptosis to eliminate virus-infected cells thereby inhibiting viral multiplication and blocking viral spread. For example, the function of the latent membrane protein I (LMPI) of EBV and the bcl-2 homologue gene A179L of African swine fever virus is to inhibit apoptosis. However, in other cases it is the virus that stimulates cell death or the host that benefits from inhibiting apoptosis, such as in fatal alphavirus encephalitis. This has been explained by assuming that virus-induced apoptosis in non-regenerating cells, for example, neurons, would be detrimental to the host.[11]
This is precisely the mechanism of pathogenesis that has been described in HIV infection. It has been shown that neuronal apoptosis occurs in the cortex of AIDS patients who were symptomatic while it was not observed in asymptomatic cases. Further, apoptosis is a mechanism of CNS injury in AIDS4 and it explains the neuronal loss and the associated AIDS dementia complex.[12]
We hypothesized that a similar mechanism could be operating in HSE. This study shows that apoptosis of neurons, glial and satellite cells does occur in HSE. The presence of neuronal loss and the evidence for apoptosis in a case of HSE suggests that apoptosis may contribute to the pathogenesis of this disease. Extensive neuronal loss could be both due to the lytic action of HSV-1 and virus induced apoptosis of the neurons. Apoptosis of the glial and satellite cells may also contribute to the severity of the disease.
Other regions of the brain were not processed for this study. It would be interesting to observe such lesions in the temporal lobes, especially the sensory cortex, since this region is often involved in HSE.
The in-vitro experiments using the neuroblastoma cell line, SNB 19, suggests that HSV-1 can induce apoptosis of cells of glial origin. Further studies employing cell lines of neuronal origin are warranted.
HSV induced apoptosis of neurons in humans has not been extensively studied. However, there are reports wherein HSV-1 has been shown to induce neuronal apoptosis in mouse brain.[13] These authors have also shown that interferon-gamma inhibits apoptosis, affording neuronal protection from destructive encephalitis during HSV-1 infection of the central nervous system.[13] It has been reported that apoptosis is induced in the spinal cord and dorsal root ganglion by HSV-2 infection, in a mouse model. Some glial cells and neurons showed evidence of apoptosis in the spinal cord while glial cells alone were apoptotic in the dorsal root ganglion.[14]
Our report has important implications in the understanding of the molecular pathogenesis of this cataclysmic disease. If further studies prove that virus induced apoptosis is indeed a mechanism of neuronal loss in HSE, it would lead to alternate therapeutic modalities in the management of this disease. Such modalities are being explored in other infections of the CNS, especially using anti-apoptogenic agents.[7]
It is encouraging to note the description of anti-apoptogenic agents like Memantine,[15] Heat shock protein (HSP) 27,[16] Caspase inhibitors,[17] Intracellular calcium chelator like BAPTA - AM,17 Inhibitor of mitochondrial calcium uptake like Ruthenium red,[17] Nitric oxide synthase (NOS) inhibitor and peroxynitrate scavenger like Uric acid[17] and Cyclosporin A.[17] These compounds may prove beneficial in preventing such neuronal degeneration. Further studies are necessary to demonstrate virus-induced apoptosis in-vitro and to detect the inhibition of apoptosis by various molecules in HSV infections of the central nervous system.

 ~ Aknowledgements Top

The authors thank Dr.Narsing A. Rao, Doheny Eye Institute, University of South California, CA, USA for providing TUNEL Kits and Mr. S.B.N. Chary for Photography. 

 ~ References Top

1.Longson M.In: Principles and practice of clinical virology, 2nd ed. Zuckerman AJ, Banatvala JE and Pattison JR., Eds (John Wiley & Sons, England) 1990; 3-42.  Back to cited text no. 1    
2.Girard S, Covderc T, Destombes J, Jheisson D, Del peyroux F, Blondel B. Polio virus induces apoptosis in the mouse central nervous system. J Virol 1999; 73: 6066-6072.  Back to cited text no. 2    
3.Marianneau P, Flamand M, Deubel V, Despres P. Induction of programmed cell death (apoptosis) by dengue virus in-vitro and in-vivo. Acta Cient Venez 1998; 49: S 13-17.  Back to cited text no. 3    
4.Shi B, De Girolami U, He J, Wang S, Lorenzo A, Busciglio J, Gabuzda D. Apoptosis induced by HIV-1 infection of central nervous system. J Clin Invest 1996; 98: 1979-1990.   Back to cited text no. 4    
5.Gavrielli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in-situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992; 119: 493-501.  Back to cited text no. 5    
6.Ren H, Wilson G. Apoptosis in the corneal epithelium. Invest Ophthalmol Vis Sci 1996; 37:1017-1025.  Back to cited text no. 6    
7.Braun JS, Novak R, Herzog KH, Bodner SM, Cleveland JL, Toumanen EI. Neuroprotection by caspase inhibitor in acute bacterial meningitis. 1999; 5: 298-302.  Back to cited text no. 7    
8.Cohen JJ. Apoptosis: Physiologic cell death. J Lab Clin Med 1994; 124: 761-765.  Back to cited text no. 8    
9.Hoffman B, Liebermann DA. Molecular controls of apoptosis: Differentiation/growth arrest primary response genes, proto-oncogenes, and tumor supressor genes as positive and negative modulators. Oncogene 1994; 9: 1807-1812.  Back to cited text no. 9    
10.Wilson SE. Stimulus-specific and cell type specific cascades: Emerging principles relating to control of apoptosis in the eye. Exp Eye Res 1999; 69: 255-266.  Back to cited text no. 10    
11.Krakaucer DC, Payne RJ. The evolution of virus induced apoptosis. Proc R Soc Lond B Biol Sci 1997; 264: 1757-62.  Back to cited text no. 11    
12.Adle-Biassette H, Levy Y, Colombel M, Poron F, Natchev S, Keohane C, Gray F. Neuronal apoptosis in HIV infection in adult. Neuropathol Appl Neurobiol 1995; 3: 218-27.  Back to cited text no. 12    
13.Geiger KD, Nash TC, Sawyer S, Krahl T, Patstone G, Reed JC, Krajewski S, Dalton D, Buchmeier MJ, Sarvetnick N. Interferon-gamma protects against herpes simplex virus type 1-mediated neuronal death. Virology 1997; 238: 189-197.  Back to cited text no. 13    
14.Ozaki N, Sugiura Y, yamamoto M, Yokoya S, Wanaka A, Nishiyama Y. Apoptois induced in the spinal cord and dorsal root ganglion by infection of herpes simplex virus type 2 in the mouse. Neurosci Lett 1997; 228: 99-102.  Back to cited text no. 14    
15.Muller WE, Schroder HC, Ushijima H, Dapper J, Borman J. gp 120 of HIV-1 induces apoptosis in rat cortical cell cultures: prevention by Memantine. Eur J Pharmacol 1992; 226: 209-14.  Back to cited text no. 15    
16.Wagstaff MJ, Collaco-Moraes Y, Smith J, de Belleroche JS, Coffin RS, Latchman D.S. Protection of neuronal cells from apoptosis by HSP 27 delivered with a herpes simplex virus based vector. J Biol Chem 1999; 274: 5061-9.  Back to cited text no. 16    
17.Kruman II, Nath A, Mattson MP. HIV-1 protein Tat induces apoptosis of hippocampal neurons by a mechanism involving caspase activation. Exp Neurol 1998; 154: 276-88.  Back to cited text no. 17    
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