ANTIVIRAL EFFICACY OF GARLIC OIL AGAINST NEWCASTLE DISEASE VIRUS

Newcastle disease is a highly contagious and devastating viral disease of poultry that distributed worldwide causing large economic losses in the poultry industry. Although vaccines are being used to control the disease, there is no effective antiviral drug used for the treatment of infections. The aim of this study is to test garlic oil for its antiviral activity against Newcastle disease virus. Garlic oil was incubated with the virus (LaSota strain) for 1 hr and 24 hrs and its antiviral effect was determined by performing hemagglutination and RT-PCR tests to detect viral surface proteins and viral genome, respectively. In addition, the toxicity of garlic oil was determined on the living organism by injecting it into chicken embryos with or without the virus. The results showed that this product played a role in the reduction of virus effectiveness through the destroying of viral surface receptors as well as the reduction of gene amplification as compared with the control group that included the treatment of the virus with a saline solution (phosphate buffer saline), which gave opposite results. In addition, there was no antiviral toxicity on the living organism since the injected embryos with the oil alone or the oil with virus were healthy and closely resemble those that have not been injected with anything. In comparison, the embryos that were injected with the virus only showed clear pathological signs that did not appear in the other groups containing the oil. These results suggest that garlic control control observed in other hand, another study has evaluated the cytotoxicity and antiviral activity against Newcastle disease virus of ivermectin, a medication used to treat many types of parasite infestations such as scabies, head lice, strongyloidiasis; and ribavirin which is an antiviral medication used to treat RSV infection, hepatitis C, Lassa fever and severe cases of influenza. The study concluded that ivermectin has strong antiviral potential at 100μg/ml and higher but some concentrations were cytotoxic while ribavirin showed strong antiviral potential at all concentrations (9) . In the current study, the concentration of garlic oil which was 50μg/ml gave good results in destroying the virus without toxicity seen in the chicken embryos. A study suggested that fucoidan from Cladosiphon okamuranus exhibited antiviral activity against Newcastle disease virus LaSota strain, and represented a potential low-toxicity antiviral compound for the poultry industry . Another study revealed that ethanolic extracts of Acacia cyanophylla leaves, Moringa peregrina leaves, Eucalyptus camaldulensis fruits and Pistacia atlantica (leaves and stem) can inhibit Newcastle disease virus completely without causing death of the chicken embryo. However, the response of the viral-infected chicken embryos was different depending on the species of the medicinal plant, plant material concentration applied and the plant part. In addition, some plant extracts showed a dose dependent relationship with the degree of the virus inhibition, whereas other plant extracts showed some toxicity on the chicken embryo (11). The results of our study revealed that the current doses of garlic oil has not shown any toxicity on chicken embryos and eliminate the virus completely, and therefore, garlic oil would be a potential antiviral drug against Newcastle disease virus.


INTRODUCTION
Newcastle disease is a highly contagious and devastating viral disease of wide range of birds, notably the domestic poultry. It is caused by Newcastle disease virus which is belonged to Paramyxoviride family. The disease has the potential to cause large economic losses in the poultry industry (1,2). It is expected that all birds are susceptible to infection, with varying of clinical signs and outcome (3) .
So far, there is no effective treatment for Newcastle disease, although secondary infections caused by bacteria may be treated by using antibiotics, so new alternative control measures are urgently required (4,5) . Recent studies have supported the effect of garlic and its extracts in a wide range of medical applications. These studies raised the possibility of revival of garlic therapeutic values in different diseases. Different compounds in garlic have been thought to have anti-tumour and anti-microbial effects. In addition, it can reduce the risk for cardiovascular diseases and also show benefit on high blood glucose concentration (6,7) . In comparison with the antibacterial effect of garlic compounds, very limited studies have been done to investigate their antiviral properties. The few studies have reported that garlic extract showed in vitro activity against influenza A and B, rhinovirus, human immunodeficiency virus, cytomegalovirus, viral pneumonia, herpes simplex virus 1, herpes simplex virus 2, and rotavirus (6) .
The aim of the present work is concerned to test garlic oil for its antiviral activity against Newcastle disease virus. The aim was achieved by evaluating garlic oil activity on destroying the outer viral protein receptors and degrading of viral nucleic acid of the virus by performing haemagglutination and RT-PCR techniques, respectively. In addition, toxicity of the oil was measured via the inoculation of embryonated chicken eggs.

Extraction of oil from garlic
Dried garlic was obtained from the supermarket, then grinded with an electric mill and then stored in glass bottles until it was used. Seventy gram of dried garlic was placed in a paper container (thimble) in the extraction apparatus (succulet) using 500ml of ethanol solvent for about 6 hrs. The solution was then placed in a rotary evaporator at 50 • C to evaporate the solvent and the remaining material was left at room temperature to dry and obtain garlic oil and then was put in a screw tube until use.

ANTIVIRAL EFFICACY OF GARLIC OIL
The role of garlic to inhibit virus effectiveness through the destroying of viral receptors or degrading viral nucleic acid was examined by performing haemagglutination and RT-PCR techniques, respectively. The concentration of garlic oil used to treat the virus was 50 µg/ml. Five groups were prepared for this purpose.
Group 1 and 2 included the incubation of garlic oil with the virus for 1 hr or 24 hrs, respectively, while group 3 and 4 represented by using garlic oil without virus (negative control 1) or PBS (negative control 2). Group 5 was represented by using virus only without garlic (positive control).

Preparation of red blood cells
Five microliters of blood were collected from chickens in vacuum tubes (EDTA.K3) and RBCs were purified as follows: The whole blood was centrifuged at 3000rpm for 5mins and the supernatant was discarded. The RBCs were suspended in PBS by mixing the tube gently. The mixture was then centrifuged for 5 mins at 2000 rpm and the supernatant was discarded. This process was repeated for 3 to 4 times until the supernatant was cleared. The RBCs were than kept at 4 • C or used directly for haemagglutination test.

Haemagglutination
The role of garlic oil to inhibit virus effectiveness through the destroying of virus receptors was examined by performing haemagglutination test. A ceramic agglutination plate which contains six wells was cleaned and prepared for this

Viral rna extraction and quantification
Viral RNA was extracted in the laboratory by using QIAamp viral RNA extraction kit (Qiagen, Germany) following the manufacturer's instructions. The concentration of the purified RNA was determined using NanoDrop spectrophotometer by UV absorption. Eluted viral RNA samples were either processed directly for RT-PCR or preserved at -20˚C until use.

Reverse transcriptase polymerase chain reaction
Viruses were detected by using two-step RT-PCR kits (Bioneer, South Korea) following the manufacturer's protocol. cDNA synthesis and PCR amplification were performed in two separated tubes using this system. The components of cDNA reaction with their volumes and final concentrations were as follows: Four microliters of template RNA were used as a starting material accompanied with 0.4 µl of forward and reverse primers (the final concentration of each primer was 0.2 pmole/ µl).
Fifteen point two microliters of nuclease-free water were then added to the reaction.
The negative control reactions were prepared either without adding primers (negative control 1) or without RNA template (negative control 2). The conditions of the first step were as follows: primer annealing at 58•C for 10 min, followed by cDNA synthesis at 42•C for 30 min and then the a denaturation step at 95•C for 5 min.
Two microliters of the synthesized cDNA was then used to perform the second step (PCR amplification). Point four microliter of forward and reverse primers were added to the reaction with 17.2 µl of nuclease-free water. The PCR conditions were as follows: initial denaturation at 95•C for 5 mins followed by 40 cycles of:

Innoculation of embryonated chicken eggs
To determine the toxicity and effectiveness of garlic oil on live organisms, fertile hen's eggs provided by Fadak company, Basrah, Iraq were inoculated with the lowest concentration of the oil (50µg/ml), which inhibited virus infectivity prior egg inoculation. The fertilized eggs were first incubated for 9 days at 37.5˚C then inoculated with the virus. Working virus concentration was prepared by diluting the stock virus 1:1000 in phosphate buffer saline containing 1% antibiotics (100 U/ mL penicillin and 100 ug/ mL streptomycin, Gibco). The volume of the inoculum was 100 µl per egg. Five groups of eggs were prepared for this purpose (10 eggs for each group). Group 1 and 2 were inoculated with a mixture of garlic oil and virus that had been incubated together for one 1 hr or 24 hrs, respectively. Group 3 and 4 were inoculated with garlic oil or virus, respectively. The eggs of group 5 were not inoculated at all. Following inoculation, the eggs were placed at 37.5˚C for 48 hrs.
The eggs were then chilled at 4˚C for 30 mins before collecting the embryos and harvesting the virus. Allantoic fluid was carefully aspirated and then clarified at 1000 xg for 5 min, and the embryos were move to Petri dishes to visualize the pathological changes.

Serological detection of virus by haemagglutination test
The role of garlic oil to inhibit virus effectiveness through the destroying of virus

Molecular detection of virus by rt-pcr
The role of garlic oil to inhibit virus effectiveness through the destroying of viral   were also normal after injection with garlic oil only and there were no haemorrhage and distortions and they were similar to the non-injected group.

Detection of virus from allantoic fluid by haemagglutination test:
Haemagglutination test was performed to detect virus growth in the allantoic fluid that was collected from the inoculated eggs with virus only, virus treated with garlic oil, and non-inoculated eggs. The results were as follows: no evidence of haemagglutination in the allantoic fluid of eggs injected with virus treated with garlic oil for 1 hr or 24 hrs. Similar results were seen in eggs injected with garlic oil only (negative control 1) and the non-injected eggs (negative control 2). In contrast, very

DISCUSSION
Garlic oil was tested as an antiviral to Newcastle disease virus, and the results were evaluated using haemagglutination and RT-PCR techniques as well as the inoculation of embryonated chicken eggs with the virus. The results revealed that garlic oil has a great ability to eliminate the virus by destroying receptors on the surface of the virus and the genetic material inside. In addition, it was found that there was no toxicity or pathological effects of this oil on the organism as confirmed by the injection of chicken embryos, and it was effectively control the virus as no viruses were identified in the allantoic fluids of all treated groups.
A recent study has considered Glycyrrhiza glabra leave extract to be a potential antiviral in vivo against Newcastle disease virus. The study showed that this extract had a role in elimination of the virus with a high survival rate of the inoculated chicken embryos (8) . This is agreed with the results of the current study as all the injected embryos were healthy and no pathological lesions were observed. On the other hand, another study has evaluated the cytotoxicity and antiviral activity against Newcastle disease virus of ivermectin, a medication used to treat many types of parasite infestations such as scabies, head lice, strongyloidiasis; and ribavirin which is an antiviral medication used to treat RSV infection, hepatitis C, Lassa fever and severe cases of influenza. The study concluded that ivermectin has strong antiviral potential at 100μg/ml and higher but some concentrations were cytotoxic while ribavirin showed strong antiviral potential at all concentrations (9) . In the current study, the concentration of garlic oil which was 50μg/ml gave good results in destroying the virus without toxicity seen in the chicken embryos.
A study suggested that fucoidan from Cladosiphon okamuranus exhibited antiviral activity against Newcastle disease virus LaSota strain, and represented a potential low-toxicity antiviral compound for the poultry industry (10) . Another study revealed that ethanolic extracts of Acacia cyanophylla leaves, Moringa peregrina leaves, Eucalyptus camaldulensis fruits and Pistacia atlantica (leaves and stem) can inhibit Newcastle disease virus completely without causing death of the chicken embryo. However, the response of the viral-infected chicken embryos was different depending on the species of the medicinal plant, plant material concentration applied and the plant part. In addition, some plant extracts showed a dose dependent relationship with the degree of the virus inhibition, whereas other plant extracts showed some toxicity on the chicken embryo (11). The results of our study revealed that the current doses of garlic oil has not shown any toxicity on chicken embryos and eliminate the virus completely, and therefore, garlic oil would be a potential antiviral drug against Newcastle disease virus.
In this study, it has been shown that garlic oil had a big role in destroying virus surface receptors and this was detected by haemagglutination test. On the other hand, some faints bands or no bands were appeared on the agarose gel after performing of RT-PCR on viruses treated with garlic oil, however, no detectible bands were seen after amplifying the virus genome taken from the allantoic fluids. The slight amplification of viral genome in the samples treated with the oil is likely to have traces of the RNA fragment of the virus where the primers have been associated with them and these fragments were amplified by RT-PCR, therefore this would not be biologically important.