Degradation of Japanese encephalitis virus by neutrophils

Abstract

The ability of neutrophils to degrade the phagocytosed Japanese encephalitis (JE) virion, via triggering of the respiratory burst and generation of toxic radicals has been investigated. JEV or JEV-induced macrophage derived factor (MDF) induces increase in intracellular oxidative signals with generation of superoxide anion (O⁻₂), via activation of cytosolic NADPH and subsequent formation of hydrogen peroxide, with maximum activity on day 7 post infection. The response was sensitive to anti-MDF antibody treatment. Further, the study revealed rapid degradation of phagocytosed JE viral protein and nucleic acid. The viral protein degradation was partially dependent on the generation of toxic oxygen species as it could be abrogated by pretreatment of the cells with staurosporine.

The recovery from viral infection is mediated by the cooperative activity of various components of the phagocyte system and different subsets of B and T effector cells (Mathur et al. 1983; Male & Roitt 1993). The neutrophil is a major phagocytic cell, which plays a central role in early host defence. The ability of neutrophils to restrict bacterial and fungal infections has long been recognized (Brummer et al. 1986), while its role in antiviral defence has been scarcely studied. Antiviral action of neutrophils may be controlled by various mechanisms, for example they may act as (i) effector cells in antibody-dependent cell cytotoxicity (ADCC) as seen in herpes simplex virus infection (Siebens et al. 1979), or (ii) release interferon-like substances in response to certain viral antigen (Rouse 1981), or (iii) degrade the virion (Van Strijp et al. 1990).

Japanese encephalitis virus (JEV), an arthropod borne flavivirus is one of the major causes of acute encephalitis in South-east Asia (Shope 1980). Peripheral neutrophil leucocytosis or infiltration of neutrophils in extraneural tissue has been reported in human and experimental animals (Grascenkov 1964; Mathur et al. 1988). During JEV infection macrophage-derived chemotactic factor (MDF) is secreted from activated macrophages which mediates its effect on neutrophils (Khanna et al. 1991). It is a pathogenesis-related 10 kD protein and plays a central role in the production of disease. It increases the capillary permeability (Khanna et al. 1994), with leakage of plasma protein, erythrocytes and cellular infiltrate in brain (Mathur et al. 1992) and lowers the serum iron levels with accumulation of iron in spleen (Bharadwaj et al. 1991). Presence of MDF has been demonstrated in the sera of Japanese encephalitis patients (Dr A. Singh, personal communication).

Interaction of neutrophils with particulate or soluble substance results in activation of both oxidative and non-oxidative mechanisms leading to generation of superoxide (O⁻₂), which dismutates to form hydrogen peroxide and other oxygen radicals, responsible for tissue damage (Weiss et al. 1978; Ward et al. 1983). We have earlier demonstrated JEV induced respiratory burst and release of the specific (vitamin B12 binding protein) and azurophilic (β-glucuronidase) granule content from neutrophils through the secretion of MDF (Khanna et al. 1993). After JEV infection, neutrophil infiltration in different organs has been shown. Therefore, the present study was planned to investigate the contribution of neutrophils towards defence against JEV. We observed that neutrophils were able to degrade phagocytosed JE virion and thus may have an important role in early antiviral defence mechanism.

Materials and methods

Animal and virus

Inbred Swiss albino mice, aged 6 weeks, obtained from this Department were used throughout the study. Japanese encephalitis virus (JEV strain 78668 A), was used as infected mouse brain suspension (Mathur et al. 1986). The infectivity titre of virus in suckling mice was 10^4.5LD₅₀/0.25 μl. It produced 100% mortality by day six following intracerebral (i.c.) inoculation while intraperitoneal (i.p.) inoculation showed no clinically evident disease. JEV-infected mouse brain suspension was used to infect BHK-21 cell monolayers. Infected cell culture fluids served as virus inoculation for all subsequent virus propagations.

Labelling and purification of the virus

To prepare (³⁵S) methionine labelled virus, BHK-21 cells infected with JEV, were incubated for 4 h in methionine free minimum essential medium (MEM)-HEPES supplemented with 0.5% FCS and then labelled for 16 h with 35 μCi of (³⁵S) methionine per ml (Bhabha Atomic Research Centre, India) in methionine free MEM-HEPES supplemented with 2% FCS. The cells were washed with saline-Tris-EDTA (STE), harvested and solubilized as described by Yasuda et al. (1990).

Virus was purified by layering 1 ml of virus suspension on 30–50% sucrose gradient followed by centrifugation at 1,00 000 g for 2 h in cold. Viral fractions were collected monitoring OD at 254 nm and fraction having high OD was collected. Purified virus was stored at −70°C in small aliquots until use. Purity of virus was also checked by electron microscopy (Philips 410 LS, The Netherlands), and it was shown that > 96% of particles were enveloped virions.

Isolation of neutrophils

The neutrophils were isolated from the peritoneal cavity of the mice 4 h after i.p. inoculation of 1 ml glycogen (0.1% in saline). The exudate was collected by flushing the peritoneal cavity with 4 ml of MEM-HEPES supplemented with 0.5 units/ml of heparin. Neutrophils were enriched by Ficoll-Hypaque gradient centrifugation. The pellet consisted of 95% purified neutrophils as checked by morphology and ability to phagocytose neutral red particles.

Preparation of macrophage derived factor (MDF)

Macrophage derived factor (MDF) has been shown to be produced by splenic macrophages of JEV primed mice (Khanna et al. 1991). Briefly, the mice were inoculated with 300 μl of 10 LD50 of JEV i.p. On day 7 p.i. the spleens were collected aseptically and splenic cells (1 × 10⁷/ml) were cultured in MEM-HEPES for 2 h at 37°C in 5% CO₂ atmosphere. The glass adherent cells were washed with phosphate buffered saline (PBS) and cultured in saline at 37°C for 24 h. The supernatant was collected, centrifuged and assayed for neutrophil chemotactic activity by modified Boyden chamber technique as described earlier by Khanna et al. (1991). The MDF was purified by high pressure liquid chromatography (HPLC) (Pharmacia, Uppsala, Sweden) on Superose-12 column using 0.1 m PBS as elution buffer. The chemotactic fractions were collected and stored at −70°C. Normal mouse spleen macrophage culture supernatant was used as control.

Indirect immunofluorescence technique

Neutrophils were spotted on Teflon coated microscopic slides and air dried. The smears were fixed in chilled acetone and examined by indirect immunofluorescence as described previously (Mathur et al. 1990). A 1:100 dilution of JE virus specific monoclonal antibody designated 98.9.5i (kindly provided by Dr E.A. Gould, U.K.) was used. The smears were screened under Dialux 20 Leitz fluorescent microscope (Leitz, Wetzlar, Germany). Results were expressed as rhe percentage of the phagocytosed cells.

Measurement of superoxide ion

Superoxide ion generation in neutrophils was measured by nitroblue tetrazolium reduction according to Baehner & Nathan (1968). Neutrophils (2 × 10⁶/400 μl PBS) collected on different days either from mice given 0.3 ml of 10 LD₅₀ JEV or control mice were treated with 0.01 m KCN, then 100 μl NBT (2 mg/ml) and 100 μl of opsonized zymosan (40 mg/ml) was added and incubated for 30 min at 37°C. The absorbance was recorded at 550 nm in a spectrophotometer. The number of millimoles of NBT-reduced was calculated using the extinction coefficient (EC) = 2.8 mm−¹ cm⁻¹ (Weiss et al. 1978). Data are presented as mean ± SE of triplicate experiments.

Assay of H₂O₂ release

H₂O₂ generation by neutrophils was determined by the method of Weiss & Ward (1982). Briefly, neutrophils (1 × 10⁶ cells/ml) were suspended in PBS with 1 mg/ml glucose and 1 mm sodium azide at pH 7.4. The reaction was initiated by the addition of 10 nm NADPH and terminated by addition of 0.1 ml of trichloroacetic acid (50% w/v) and centrifuged. To 1 ml of supernatant 0.2 ml of 10 mm ferrous ammonium sulphate and 0.1 ml of 2.5 m potassium thiocynate was added. The absorption of the red ferrithiocynate complex was measured at 350 nm spectrophotometrically and was compared with the standard curve generated from dilutions of the reference solution of H₂O₂. Results were expressed as nM of H₂O₂released/min/10⁶ cells using an extinction coefficient (EC) = 81 m⁻¹ cm⁻¹. 10⁻⁷ m n-formyl-methionyl-leucyl-phenylalanine (FMLP) was used as positive control.

Myeloperoxidase assay

Enriched neutrophils (1 × 10⁷cells/0.1 ml) were homogenized in five volumes of ice cold 50 mm phosphate buffer, pH 6.0 containing 0.5% hexadecyl-trimethyl-ammonium bromide. The homogenate was centrifuged at 40 000 g for 10 min at 4°C and myeloperoxidase (MPO) activity was determined in the supernatants spectrophotometrically at 460 nm over 2 min (Goldblum et al. 1985).

NADPH oxidase assay

The oxidase was obtained from MDF activated or normal neutrophils as described by Markert et al. (1984). Briefly, 2 ml of opsonized zymosan (22.5 mg/ml) was mixed with prewarmed purified neutrophils (1.5 × 10⁸ cells/ml) and incubated at 37°C for 7 min. Reaction was stopped by adding 4 ml of ice cold Ca²⁺ and Mg²⁺ free PBS and centrifuged at 4°C for 5 min at 400 g. The cells were pelleted in ice cold 0.35 m sucrose containing 0.5 mm phenyl methyl sulphonyl fluoride (PMSF). The cells were disrupted in a sonicator (w-380, Ultrasonics Inc. New York) and centrifuged at 250 g for 10 min at 4°C and the supernatant was collected. The membrane fraction containing the oxidase was pelleted by centrifugation at 100000 g for 30 min and resuspended in 0.34 m sucrose at a protein concentration of 1 mg/ml. The cell free NADPH-oxidase activity was assayed in the presence and absence of MDF. Results were expressed as nM cytochrome C reduced/min/1.5 × 10⁸ cells.

Virus degradation assay

The degradation of JEV glycoprotein was studied after incubating the (³⁵S) methionine labelled virus with neutrophils (3 × 10⁷ cells/ml) at 37°C for 1 h. The cells were washed thrice with PBS and were resuspended in 120 μl PBS with foetal calf serum, then incubated at 37°C for different periods in a shaking water bath and cooled on ice. The cells were solubilized in 500 μl of 1% Triton X (TX)-100 for 30 min and layered onto 500 μl 6% sucrose at 37°C, then pelleted through sucrose at 12 000 g for 3 min. The TX pellet and two 200 μl samples from the top supernatant were mixed with scintillation fluid and counted in the liquid scintillation β-counter (Van Strijp et al. 1990). Results were expressed as:

SDS-PAGE of viral proteins

SDS-PAGE was performed in 10% gels (Laemmli 1970) to confirm the viral protein degradation. The gel was stained in Coomassie brilliant blue overnight and then destained to visualize the protein bands.

Degradation of viral RNA

Degradation of (³H)-Uridine labelled viral RNA in neutrophils was studied by measuring the TCA precipitable radioactivity in a liquid scintillation β counter. Radiolabelling of viral RNA was carried out by the technique of Stollar et al. (1966) using (³H)-Uridine (14 μCi/ml; Bhabha Atomic Research Centre, India). Neutrophils (3 × 10⁷/ml) were incubated with (³H)-Uridine labelled virions at 37°C for 60 min, washed three times and lysed or further incubated for 120 min. These cells were suspended in water, frozen and thawed. To this, 10% TCA (1:1) was added and incubated for 30 min at 4°C, centrifuged for 10 min at 12 000 g and washed with 5% TCA. The pellet was harvested onto fibreglass filter. The radioactivity was measured in LKB-Wallac Beta Counter (Pharmacia, Uppsala, Sweden). The cpm in controls and test were calculated by substracting background cpm. The experiments were performed in triplicate and their results were presented as mean ± SE.

Results

Phagocytosis of JE virus

The phagocytic activity of normal neutrophils, exposed to purified JEV and foetal calf serum in vitro for different time was studied. The phagocytosed virus was detected by indirect immunofluorescence using JEV specific monoclonal antibody.

Findings summarized in Figure 1 show that maximum uptake of virus in neutrophils occurred at 1 h. The infected cells showed a bright cytoplasmic fluorescence (Figure 2). Control cells were given normal mouse brain suspension and did not show fluorescence.

Figure 1.

Demonstration of number of JEV positive immunofluorescent cells at different time periods after JEV (▪) or costimulation with MDF and JEV (). Control cells (C) were stimulated either with MDF (□) or normal macrophage culture supernatant (*). IF positive cells were counted in 100 high power fields (hpf). Values are presented as mean ± SE of triplicate experiments.

Figure 2.

JE virus antigen in a neutrophil after 60 min of incubation, stained by indirect immunofluorescent technique. Magnification ×250.

Effect of JEV on NADPH activity of neutrophils

Enhancement of the specific activity of NADPH oxidase in peritoneal neutrophils (1.5 × 10⁸/ml), stimulated in vitro with JEV is illustrated in Table 1. The NADPH oxidase activity in terms of superoxide dismutase-inhibitable cytochrome C reduction was significantly higher in JEV treated cells as compared to control.

Table 1.

Effect of JEV and MDF on NADPH oxidase activity of neutrophils

NADPH oxidase activity in MDF activated or nonactivated neutrophils after 1 h of in vitro JEV stimulation. Cytochrome C reduction was measured spectrophotometrically in cell supernatants at 550 nm. Values are presented as mean ± SE of five experiments. † P < 0.05 * P < 0.0001.

Respiratory burst response

As we have earlier demostrated that JEV infected neutrophils released reactive oxygen metabolites in vivo (Khanna et al. 1993), we were curious to see whether superoxide production also occurs with JEV in vitro. Thus, normal neutrophils were treated with JEV and NBT reduction was evaluated. The results showed that JEV triggered significant (P < 0.05) superoxide generation at 30 min post stimulation (mean value = 0.20 ± 0.09 nm/min/10⁶ cells). The NBT reduction observed in neutrophils stimulated with normal mouse brain suspension was included as control (mean value = 0.11 ± 0.003 nm/min/10⁶ cells).

Hydrogen peroxide release in neutrophils

The present experiment was carried out to ascertain time course of release of H₂O₂ in neutrophils derived from mice inoculated with 10 LD₅₀ of JEV i.p. and from controls which were given normal mouse brain suspension i.p. The data presented in Table 2 show that H₂O₂ release in neutrophils reached a peak from day 7–9 following JEV infection and then gradually declined. Therefore, in further experiments observations were recorded at day 7 p.i. The H₂O₂ release showed a progressive increase with an increasing number (1 × 10⁴/ml to 1 × 10⁷/ml) of neutrophils.

Table 2.

JEV-induced H₂O₂ release in neutrophils

JEV induced H₂O₂ release at different days after intraperitoneal inoculation of JEV. Control mice were given normal mouse brain suspension. The results are mean ± SE of five experiments.

Abrogation of H₂O₂ production by JEV specific antisera treatment

Groups of mice were injected i.v. with JEV specific antiserum (1 : 10 dil.) and were challenged 24 h later with 10 LD₅₀ of JEV i.p. Peritoneal neutrophils collected on day 7 p.i. were assayed for H₂O₂production. The findings showed that pretreatment of mice with antisera completely abrogated the increase in H₂O₂ production (mean value = 1.01 ± 0.003 nm/min/10⁶ cells).

Release of myeloperoxidase

The release of MPO by neutrophils (1 × 10⁷/ml) collected on different days from mice inoculated with 10 LD₅₀ of JEV intraperitoneally was measured by hexadecyl-trimethyl ammonium bromide, reduction assay. The findings summarized in Table 3 show that maximum release of MPO was observed from day 7–9 p.i.

Table 3.

Myeloperoxidase release by neutrophils after JEV infection

* P < 0.05. Mice were injected with JEV i.p. and neutrophils collected on different days were assayed for the release of MPO. Neutrophils from normal mice were collected on respective days and were taken as control. Each value represents mean ± SE of five experiments.

Effect of MDF on phagocytosis

We have earlier reported production of pathogenesis related macrophage derived factor (MDF), a low molecular weight neutrophil chemotactic polypeptide during JEV infection in mice (Khanna et al. 1991). Therefore, we investigated whether MDF facilitates the uptake of virus. Neutrophils (3 × 10⁷/ml) were stimulated first with 5 μg of MDF for 2 min followed by purified JEV in vitro. Findings presented in Figure 1 show that MDF stimulation increased the phagocytosis of virus by neutrophils at 60 min as compared to JEV alone. Control neutrophils were stimulated with MDF alone for 60 min

Modulation of respiratory burst by MDF

Normal purified neutrophils (1.5 × 10⁸ cells/ml) were stimulated with 5 μg MDF or prestimulated with MDF followed by JEV and their NADPH oxidase activity was evaluated in vitro. Findings summarized in Table 1 show that MDF alone or in combination with JEV induced significantly high NADPH oxidase activity as compared to control.

In another set of experiments, neutrophils (2 × 10⁶ cells/400 μl PBS) were stimulated in vitro with 5 μg of MDF and superoxide generation was evaluated. Neutrophils treated with MDF elicits rapid (maximum at 30 s) respiratory burst response (mean value = 0.23 ± 0.08 nm/min/10⁶ cells; P < 0.05) as compared to control (mean value = 0.12 ± 0.001 nm/min/10⁶ cells).

MDF as a inducer of H₂O₂ release

The time course of peak H₂O₂ release by neutrophils in response to in vitro stimulation with MDF (5 μg) was studied. The findings showed that MDF elicited rapid and transient response, with peak H₂O₂ production at one min. (4.67 ± 0.47 nm/min/10⁶ cells).

The effect of MDF-specific antisera was investigated on H₂O₂ production. Anti-MDF antisera (1:10 dilution) was inoculated i.v. into mice followed 24 h later by 10 μg of MDF i.v. Peritoneal neutrophils were assayed for H₂O₂ production. A reduction in H₂O₂ release was observed in anti-MDF antisera treated mice (mean value = 2.001 ± 0.024 nm/min/10⁶ cells) as compared to MDF inoculated mice (mean value = 5.01 ± 0.66 nm/min/10⁶ cells).

In another set of experiment, the response to JEV was partially inhibited by pretreatment of mice with anti-MDF antibody (1:10 diluted) (mean value = 1.98 ± 0.032 nm/min/10⁶ cells) as against JEV-induced H₂O₂ release in neutrophils (mean value = 4.55 ± 0.89 nm/min/10⁶ cells). Normal mouse serum had no effect on H₂O₂ production mediated by JEV (mean value = 4.7 ± 0.56 nm/min/10⁶ cells).

Effect of staurosporine on H₂O₂ release

The effect on H₂O₂ release by MDF stimulated neutrophils which were pretreated with protein kinase C inhibitor, staurosporine, was studied. The neutrophils were incubated in vitro with different concentrations of staurosporine for 2 min followed by stimulation with 5 μg MDF. Progressive inhibition of H₂O₂ production was observed with increasing concentration of staurosporine (Table 4).

Table 4.

Effect of staurosporine on H₂O₂ release

Neutrophils (10⁶/ml) were incubated with different concentrations of staurosporine for 2 min and stimulated with 5 μg MDF. Control cells were stimulated with normal splenic macrophage culture supernatant. The results are mean ± SE of five experiments.

Release of myeloperoxidase by MDF

Release of MPO by neutrophils in response to in vitro stimulation with MDF (5 μg) was measured. The findings showed that release of MPO was higher with MDF (A₄₆₀ = 0.041 ± 0.002/10⁷cells) than in neutrophils stimulated with normal mouse macrophage culture supernatant (A₄₆₀ = 0.02 ± 0.015/10⁷ cells).

Time course of viral protein degradation

In studying degradation of viral glycoprotein, neutrophils (3 × 10⁷ cells/ml) were incubated with 30 μl of purified (³⁵S)-Methionine labelled virus in presence of serum for 1 h, treated with TX-100 and layered onto 6% (w/v) sucrose. The time dependent degradation of the viral protein in the neutrophils was measured and compared with MDF prestimulated and similarly treated neutrophils. The findings summarized in Figure 3 show that significant degradation of viral protein occurred at 120 min (44 ± 5%, P < 0.05). The percentage degradation at the same time was higher (62 ± 3%) in cells prestimulated with MDF. However, a partial inhibition of the viral protein degradation enhanced by MDF was observed following pretreatment of cells with 40 nm of staurosporine. To confirm viral protein degradation SDS-PAGE was performed. The cells were solubilized after 120 min and electrophoresed on 10% polyacrylamide gel, which showed presence of low molecular weight proteins as compared to the normal JEV protein pattern (Figure 4).

Figure 3.

Virus degradation in neutrophils. (○) JEV only; (▪) cells pretreated with MDF; (•) cells pretreated with staurosporine and MDF. Neutrophils were assayed in their ability to degrade viral protein at different time periods. 3 × 10⁷ cells/60 μl were incubated with (³⁵S) labelled virus for 1 h at 37°C. % degradation was measured at different time periods as follows: % degradation = cpm in supernatant ×100/cpm in pellet + supernatant. Each value represents mean ± SE of three experiments.

Figure 4.

SDS-PAGE of viral protein after phagocytosis by PMN. Lane 1, M_r markers; Lane 2, purified JEV protein pattern; Lane 3, after 120 min phagocytosis by PMN.

Degradation of viral RNA

For studying the degradation of viral RNA the neutrophils (3 × 10⁷ cells/ml) were incubated with (³H)-Uridine labelled virion and TCA precipitable radioactivity was measured 60 and 120 min later. Findings summarized in Table 5 show substantial viral RNA damage at 60 min in neutrophils. The percentage viral degradation was significantly higher at 120 min of incubation (P < 0.05).

Table 5.

Degradation of viral RNA by neutrophils at different time intervals

Degradation of viral RNA which was phagocytosed by neutrophils for different time periods. Results shown are TCA-soluble cpm as a percentage of the total associated cpm ± SE of triplicate experiments. The control values represnt degradation of virus incubated in MEM alone at respective time periods. 0.069 ± 0.027.

Discussion

During JEV infection peripheral neutrophil leucocytosis along with inflammatory cell infiltration in various tissue has been observed (Johnson et al. 1985; Mathur et al. 1988). Our previous studies have shown that this effect is mediated by the secretion of a macrophage derived neutrophil chemotactic factor (Khanna et al. 1991) with a plethora of biological effects. In the present study, we have attempted to investigate the role of phagocytic cells in anti-viral defence. Our data show the modulating effect of MDF on JEV induced reactive oxygen metabolite (ROM) production by the peritoneal neutrophils which could be blocked by pretreatment with anti-MDF antibodies, indicating that MDF may be responsible for the production of ROM. We further demonstrated that stimulation with MDF results in enhanced degradation of phagocytosed viral protein in neutrophils. The degradation of virion was partially dependent on the toxic oxygen species generation in neutrophil as degradation of viral protein elicited by MDF was partially abrogated by prior treatment of neutrophils with staurosporine.

In a number of viral, bacterial and fungal infections neutrophils contribute significantly in defence and provide protection (Tsuru et al. 1981; Van Strijp et al. 1990), while in others it resists killing of microorganisms (Riley & Robertson 1984). Both oxidative and non-oxidative mechanisms have been employed by neutrophils for the digestion of the invading microbe. The present study revealed an increase in intracellular oxidative signals of neutrophil following phagocytosis of particulate (Virus) or soluble (MDF) stimulants with subsequent release of superoxide anion (O⁻₂) by activation of NADPH. The transduction sequence causing activation of NADPH in neutrophils is likely to involve an enhancement of cytosolic calcium (Srivastav et al. 1994) and activation of protein kinase C (Khanna et al. 1993). Present study shows the production of H₂O₂ with concomitant release of myeloperoxidase which peaked on day 7 after JEV inoculation coinciding with the maximum production of MDF. MPO has been shown to convert H₂O₂ to toxic oxygen metabolites and hypochlorous acid (Weiss 1989), which play an important role in tissue damage. MDF showed significantly increased capacity for induction of respiratory burst and appears to be the central mediator for production of oxygen metabolites of neutrophils during JEV infection.

While this succession of processes helps to explain the formation of reactive oxygen metabolites in neutrophil and perivascular environment, the question remains whether the phagocytosed virus is actually degraded by the neutrophil. The findings of the present study demonstrate a rapid degradation of phagocytosed viral protein and nucleic acid by neutrophils. The degradation was enhanced by pretreatment of neutrophils with MDF. In JEV infection one of the mechanisms of early killing appears to be reactive oxygen metabolites mediated as the viral degradation was partially abrogated by staurosporine, a potent inhibitor of protein kinase C. However, not all the particles are degraded via this mechanism. Those virions which evade engulfment or killing by neutrophils could be taken up by macrophages or T lymphocytes (Mathur et al. 1986, 1988), followed by establishment of latent infection as observed in JE patients (Sharma et al. 1991) as well as in mice (Mathur et al. 1986). Several observations have been made showing that toxic oxygen radicals generated during viral infection are involved in killing of organism (Belding et al. 1970), while Van Strijp et al. (1990) have shown no role in herpes simplex viral protein degradation.

The data indicate that neutrophils on stimulation with JEV or MDF generate reactive oxygen metabolites and help in degradation of the phagocytosed virus which may be one of the early defence mechanisms for killing of the virus.