In vitro Antiviral Activity of Nigella sativa against Peste des Petits Ruminants (PPR) Virus

Kiran Aqil1, Muti-ur-Rehman Khan2, Asim Aslam2, Aqeel Javeed5, Rizwan Qayyum1, Farooq Yousaf,1 Farkhanda Yasmeen1, Muhammad Luqman Sohail3 and Sajid Umar4,*

1Veterinary Research Institute, Lahore 54000

2Department of Pathology, University of Veterinary and animal Sciences, Lahore 54000

3University College of Veterinary and Animal Sciences, The Islamia University of Bhawalpur, Bhawalpur 63100

4Department of Pathobiology, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi 46000

5Department of Pharmacology, University of Veterinary and animal Sciences, Lahore 54000

ABSTRACT

Peste des petits ruminants (PPR) is an important viral disease of sheep, goat and other small ruminants, imparting substantial economic losses. In current study, antiviral activity of six dilutions of Nigella sativa (N. sativa) alcoholic extracts against the PPRV was investigated in vitro. Vero cell lines infected with PPRV were treated with six dilutions alcoholic extracts of N. sativa (200, 100, 50, 25, 12.5 and 6.25 µg/mL). The antiviral effects were judged by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide colorimetric (MTT) assay. Plaque reduction assay was used to test the mode of action. Out of six dilutions, three dilutions (50, 25, 12.5 µg/ml) showed significant antiviral activity and cell survival was more than 50%. Two dilutions of N. sativa (200 µg/ml and 100 µg/ml) extracts showed cytotoxicity to Vero cell lines. In the plaque reduction assay, alcoholic extracts of N. sativa (50 µg/ml) significantly reduced the plaques count as compared with the control negative (P<0.05) in all the tested modes of action. The current experiment revealed that the alcoholic extracts of N. sativa (50 µg/ml) could increase the viability of infected cells, and curtail the cytopathic effects of PPRV. N. sativa seed extracts should be explored in field conditions for the control of PPR.

Article Information

Received 22 April 2017

Revised 23 June 2017

Accepted 07 July 2017

Available online 05 October 2018

Authors’ Contribution

KA, MuR, AA and AJ designed and planned the study. KA, RQ, FY and FY executed the experiments and provided technical help. SU and MLS performed statistical analysis of the data and wrote the article.

Key words

Peste des petits ruminants virus, N. sativa, Antiviral activity.

DOI: http://dx.doi.org/10.17582/journal.pjz/2018.50.6.2223.2228

* Corresponding author: sajncvi@gmail.com

0030-9923/2018/0006-2223 $ 9.00/0

Copyright 2018 Zoological Society of Pakistan

Introduction

Peste des petits ruminants (PPR), also called as kata, goat plague, syndrome of stomatitis, pneumoenteritis and ovine rinderpest. It is a viral disease of both domesticated and wild small ruminants. It imparts great threats to food security and livelihood of livestock farmers in Middle East, Asia and Africa (Banyard et al., 2010). The infection is characterized by fever, pneumonia, discharge from oral cavity and nostrils, ulceration of the mucous membranes and inflammation reaction at the gastrointestinal tract, causing diarrhoea. This disease has been reported to be an emerging infection in various regions around the globe (Banyard et al., 2014). The etiological agent is Peste des Petits Ruminants Virus (PPRV) of genus Morbillivirus and family Paramyxoviridae. A genome size of 15948 bp makes it the largest member of this Morbillivirus (Bailey et al., 2005). Its genome codes for six proteins i.e. nucleoprotein (N), viral RNA-dependent polymerase (L), RNA-polymerase phosphoprotein co-factor (P), matrix protein (M), fusion protein (F) and hemagglutinin protein (H) (Diallo, 2003). It is transmitted through direct contact with already infected animals through secretions and fecal material (Truong et al., 2014). Although, only a single serotype of PPRV is reported to exist, phylogenetic analysis indicated that viral strains can be divided into four lineages. Out of which three occur in Africa only, while one exists in Africa and Asia (Parida et al., 2015). There is no standard medications protocol exists for the treatment of the disease, but there are vaccines (Abubakar et al., 2011).

Currently, feed industry is focusing on various substitutes for antiviral drugs (Al-Mufarrej, 2014). Antimicrobial agents of plant origin such as essential oils, plants extracts, and complete plant substances are considered as alternatives to the traditional antimicrobial feed additives. Nigella sativa are one of such alternatives that could be used as feed additives in order to reduce the pathogen load in animals. It has been reported that N. sativa or its constituents such as thymoquinone, nigellone, and thymohydroquinone demonstrated several pharmacological activities such as anti-inflammatory, anti-ischemic, antioxidant and antibacterial,antiviral antihistaminic, hepatoprotective and immunopotentiating effects (Hosseinzadeh et al., 2012, 2013). Thymoquinone has been found as the key bioactive component of N. sativa seeds. Recently, clinical and experimental studies have demonstrated many therapeutic effects of Thymoquinone including immunomodulatory, anti-inflammatory, anti-tumor, and antimicrobial effects (Ahmad et al., 2013; Azeem et al., 2014; Al-Mufarrej, 2014). Large number of biological targets and nearly no adverse side effects make N. sativa a key player for the cure of immunosuppressive diseases. The goal of this communication is to analyze the antiviral potential of various dilutions of alcoholic extract of the N. sativa.

 

Materials and Methods

Cells and viruses

Vero cell lines were propagated in Dulbecco’s minimal essential medium (DMEM; Gibco-BRL, Grand Island, NY) enriched with 10% calf serum (CS; Gibco-BRL) and antibiotics. Vero cells were seeded at the rate of 104 cells/well on 96 wells cell culture plates and were incubated at 37°C in humid atmosphere having 5% CO2. Cells were counted by haemocytometer (Ali et al., 2017). After 48-72 h of incubation, the cell lines were examined through inverted microscope. The 80% to 90% confluent cell lines in each well was picked for further study.

Purified PPRV virulent strain was provided by Dr. Muhammad Anees (Veterinary Research Institute, Lahore, Pakistan). This virus was originally isolated from field samples and characterized using molecular techniques. Virus was propagated on Vero cell lines by subsequent passages. TCID50 was measured using the statistic analysis by Reed and Muench method (Gialleonardo et al., 2011). PPRV with a titer of 103 TCID50/50µl was used for antiviral and cytotoxicity activity.

Preparation N. sativa extracts

NS seeds were procured from herbal store in Lahore, Pakistan. The taxonomic identification of the plant seeds was done at the University of the Punjab, Lahore, Pakistan. The seeds were washed, dried and grounded into a coarse powder, and then weighed by analytical balance. Powdered seeds were extracted with 95% ethanol using soxhlet apparatus. After reduced pressure evaporation, dried powder was dissolved in 20mL sterilized water and filtered by a 0.22 μm millipore filter to remove bacteria (5.12 g/20mL of pure water). Extract was then dissolved in 2 mL media to obtain two 200 µg/mL dilutions for antiviral and cytotoxicity assay. Serial two fold dilution of 200 µg/mL was done to obtain 100, 50, 25, 12.5 and 6.25 µg/mL in duplicate manner. Sterility of extract was checked on Nutrient, Maconkey and Sabroud agar (Ulasli et al., 2014).

Antiviral activity assay

A suspension (50μL) having 103 median tissue culture infectious doses (TCID50) virus were added to vero cells monolayer in 96 well plates and were incubated at 4°C for 60 min to permit attachment. After that unabsorbed virus was removed by aspirating the medium. After washing the cell monolayers with PBS, six dilutions of the test samples (200, 100, 50, 25, 12.5 and 6.25 µg/mL) were added in the plates. Infected cells without treatment and uninfected cells were kept as controls. The experiment was executed in triplicate with infected- untreated and uninfected-treated as controls. All the plates were incubated at 37°C in humidified 5% CO2 and were observed daily by inverted microscope. Scoring of viral cytopathic effects (CPE) of the tests was performed under light microscopy in comparison to the virus control and cell control. Cell viability was observed by by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide colorimetric (MTT) method assay (MTT colorimetric assay) for evaluation of antiviral activity as described previously (Bhanuprakash et al., 2007; Xu et al., 2012).

Cytotoxicity assay

The cytotoxic effects of extracts on Vero cell monolayers were evaluated by quantifying the viability of cell through MTT assay (Li et al., 2005). Briefly, Vero cell lines were grown into monolayer in 96-well plates and the medium was eliminated. One mL test sample with various concentration (suspension made in MEM supplemented with 2% CS) was poured in each well. The dilution medium was kept as control. This experiment was conducted in triplicate, and plates were incubated at 37°C in a humidified 5% CO2. After 96 h, MTT solution (20 μL, 5 mg/mL in PBS) was added in each well and the plate was then again incubated for 4 h for MTT formazan formation. After removal of the medium, formazan crystals were dissolved using 100 μL dimethylsulfoxide. After 20 min, the contents were homogenized using microplate shaker. The optical densities (OD) were read at wavelengths of 490 nm through spectrophotometer. Cells were observed under inverted microscope for visible morphological changes and compared with controls. Maximum non-toxic concentration (MNTC) was calculated on the basis of cellular morphological changes in Vero cells (Bhanuprakash et al., 2007; Xu et al., 2012).

 

Plaque reduction assay

The inhibitory effects of the extracts on the PPRV replication in vero cells were analyzed through plaque reduction assay, performed as described previously (Xu et al., 2012). The tests for the plaque reduction assay were performed in 24 well plates.

Virus inactivation assay

A concentrated virus suspension along with equal volumes of extract dilutions were incubated at 37°C for 60 min. The mixture alone, without any addition of test sample was kept as control. The remaining virus in the sample was diluted (100 fold) before placing on monolayers for 60 min. Cell monolayers were washed with PBS before overlaying with DMEM (2% CS and 1% methylcellulose). After 5 days of incubation, the cells were stained using 0.1% crystal violet in 20% methanol and plaques were enumerated after washing.

Inhibition of virus attachment

Extract dilution and virus suspension were placed on monolayers to allow virus to adsorb for 60 min at 37°C, with and without test samples. After that unabsorbed mixture was removed. Adsorption efficiency was determined by counting plaques as described for virus inactivation assay.

Intracellular inhibition assay

The cell monolayer were infected with virus and incubated at 37C for 1 h. The unabsorbed virus was removed by aspirating the medium. Before overlaying the cell monolayers extract dilutions, washing was done by PBS. The wells overlaid with DMEM without the addition of test sample were kept as control. The plate was incubated at 37°C for 120 min. Plaque reduction assay was done similar to the test above.

Pretreatment assay

Different dilutions of extract were added in cell monolayers and were set to be incubated at 37°C for 2 h. Washing of cell monolayers were done by PBS and overlaid virus. The plate was then incubated at 37°C for 60 min. The cell monolayers without test samples were kept as control. Plaque reduction assay were carried out as described for Inhibition of virus attachment.

Statistical analysis

Statistical analysis was performed using the Statistical Product and Service Solutions (version 17.0, SPSS Inc., Chicago, IL, USA). Data are shown as mean± SD and Duncan’s multiple range tests were conducted to separate means.

 

Results

Cell cytotoxicity and antiviral activity of the extracts

N. sativa extract dilutions (200 and 100 μg/mL) showed high cytotoxicity to Vero cells and low anti-PPRV activity. The MTT reduction assay indicated that the four extract dilutions of N. sativa (50, 25, 12.5 and 6.5 μg/mL) showed no cytotoxic effects on Vero cells. The CPE reduction assay indicated that the 50μg/mL yielded significant (P<0.05) antiviral activity. The MNTC value of the extract confirmed that cytotoxicity did not influenced the anti-PPRV activity of extracts. The changes in morphology of infected Vero cells infected with PPRV and treated with different dilutions of N. sativa were shown in Figure 1. The cells were dense and fine in normal Vero cells after humidified incubation (5% CO2) at 37°C. The PPRV infected cells showed CPE on the whole monolayer of cells at about 72 h postinfection (Fig. 1A). The dense bundles of fibers, with an intact structure and well-defined border with few CPE, indicated that 50 μg/mL and 25 μg/mL could inhibit CPE formation caused by PPRV (Fig. 1B, C). Moreover, extract dilution 50 μg/mL showed maximum percentage of survival cell followed by 25 μg/mL 12.5 μg/mL and 6.5 μg/mL (Fig. 2).

 

Table I.- Number of plaques in different plaque reduction assays.

Plaque reduction assay	Number of plaques (Mean±SD)
Virus inactivation	22.6a ±2.3
Inhibition of virus attachment	20.9a±1.6
Intracellular inhibition	13.bb ±0.89
Pretreatment	15.4b ±1.5
Negative control	36.8c ±0.78

a-c, different lowercase letters represent significant differences among groups (P < 0.05). The 50µg/ml N/sativa extract was used in plaque forming assay. The study was performed in triplicate.

 

Plaque reduction assay

N. sativa dilution (50μg/mL) was used in the mode of action study. The numbers of plaques under the various modes of action are shown in Table I. Numbers of plaques in all plaque reduction Assays differed significantly from the negative control (P < 0.05). Out of all the assays, intracellular inhibition assay yielded the best antiviral activity. Number of plaques had statistically significant difference (P < 0.05) from the virus inactivation assay and inhibition of virus attachment assay.

 

Discussion

Peste des Petits Ruminants (PPR) is a serious disease of sheep and goat causing huge economic losses every year. As PPR is a viral disease, there exists no particular treatment for the disease and post-exposure therapeutic approaches for infection are not described much in the literature (Balamurugan et al., 2014). The antiviral activities of N. sativa seeds against several kinds of virus were reported in previous literatures (Dorucu et al., 2009; Shewita and Taha, 2011; Ahmad et al., 2013; Umar et al., 2015, 2016). Numerous studies reports the use of N. sativa as liver tonics, anti-diarrheal, analgesics, and anti-bacterial. Recently, clinical and experimental studies have demonstrated many therapeutic effects of thymoquinone including immunomodulatory, anti-inflammatory, anti-tumor, and antimicrobial (Al-Mufarrej, 2014). Due to a number of different biological targets and no side effects, NS has attained the potential therapeutic interest to cure viral diseases involving immunosuppression (Umar et al., 2016). The result of the antiviral activity of extracts from N. sativa against PPRV showed N. sativa extracts possessed the antiviral activity according the viability of infected cell assay. Antiviral activity assay results showed that alcoholic dilutions of N. sativa 200, 100, 50, 25, 12.5 and 6.25 µg/ml showed 9.4%,7.4%, 80.4%, 77.7%, 75.3% and 59.8% cell survival percentage, respectively. More than 50% cell survival percentage of Vero cells occurred in four dilutions after virus infection. Similarly, alcoholic dilutions of N. sativa (200, 100, 50, 25, 12.5 and 6.25 µg/ml) showed 20%, 33.4%, 93.6%, 90.7%, 88.9% and 89.6 % cell survival percentage respectively for cytotoxic activity assay. Alcholic extract of N. sativa showed toxicity at 200 and 100 µg/ml to Vero cells. Extracts of medicinal plants showed antiviral activity against goat pox virus (Bhanuperkash et al., 2008). In another study, N. sativa showed significant antiviral activity against Murine cytomegalo virus (MCMV) (Salem and Hossain, 2000) as compared to controls. It can be assumed that N. sativa seed may contain active compounds that would be effective in treatment of PPR virus infections in goats.

To study the mode of action of 50 μg/mL N. sativa extracts, a sequence of plaque reduction experiments were done in the current study, including inhibition of virus attachment assay, virus inactivation assay, intracellular inhibition assay and pretreatment assay (Li et al., 2005; Harden et al., 2009). The decrease in plaques count per well denoted the antiviral activity of the test dilution of N. sativa extract. The findings of all these plaque reduction procedures revealed that 50 μg/mL of N. sativa extracts significantly reduced the number of plaques caused by PPRV in all the four modes of actions as compared to the negative control. Results revealed that the plaques in intracellular inhibition assay and pretreatment assay were significantly lesser than the rest of the test groups. It can be attributed to the ability of test drug to pass through cell membranes to enter into other cells, and protects them from the virus induced injury and inhibits the viral activity more effectively with the assistance of cells. The results of this study suggested that N. sativa seed extracts are a potential source of antiviral materials effective in controlling PPRV in vitro. N. sativa seed extracts should also be studied in field experiments as a control measure for PPR. It may be suggested that N. sativa seed extracts posses anti-PPRV activity and play a significant role in diminishing the pathogenic effects of PPRV in goats. This study strengthens our belief that N. sativa seed extracts (50 μg/mL) is safe and useful antiviral agent against PPRV.

 

Acknowledgements

We are grateful to Dr. Muhammad Anees (Veterinary Research Institute, Lahore, Pakistan) for providing virus and technical help for this study

 

Statement of conflict of interest

None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.

 

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