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Olive Leaf Extract (OLE) exhibits antiviral activity against H5N1 Avian Influenza
virus
Saif, M.A*; Arafa, A.*; Madbouly, H.M**
* Animal Health Research Institute, Egypt
** Faculty of Veterinary Medicine, Beni Suef University, Egypt
Abstract
In this study, the antiviral activity of Olive leaf extract (OLE) was explored
against the highly pathogenic avian influenza H5N1 Egyptian virus. Invitro and Invivo
studies were done to explore the effect of OLE against virus growth and replication.
Firstly, the cytotoxicity assay was done to detect the highest safe dose concentration to be
used for antiviral studies, using different dilutions of virus on MDCK cell lines.
Cytopathic inhibition assay was done for 4 trials which named as(A, B, C & D);
Trial A: MDCK cells infected with H5N1 virus then treated with OLE after one hour of
incubation, Trial B: MDCK cells treated with OLE then infected with H5N1 virus after
one hour of incubation, Trial C: MDCK cells infected with H5N1 virus and treated with
OLE at the same time (competitive assay), Trial D: MDCK cells treated with a mixture of
H5N1 virus and OLE incubated for one hour at 37°C.
Results revealed that the treated MDCK cells (Trial B) with either watery or
alcoholic OLE showed no CPE in comparison to the non-treated cells. The viral titer
only decreased by 1 to 2 logs in cells infected by AI virus then treated with either
watery and/or Alcoholic OLE(Trial A). In the competitive assay, (Trial C) where the viral
infection and OLE treatment were done at the same time, the TCID was significantly
lowered (10-8.68 & 10-7.87) in both watery and alcoholic extracts respectively in
comparison to non-treated cells (10-14.25). While in Trial D, to detect the direct effect of
the extracts on the virus replication, the incubated mixture for one hour was used and the
results obtained revealed that the virus titer decreased by 2 logs only in comparison to the
non treated cells.
Data obtained from plaque reduction assay also showed mostly the same results. In vivo
experimental work, The use of OLE 3days pre- and 3 days post-infection, protects 70 %
of birds ( Group 1) while group 2 in which the OLE is only given post infection also
protect 70 % of birds. But the difference appeared clinically by the delayed beginning of
mortalities in case of pre-infection treatment. This clinical result supported by the results
of group 3 in which the birds prophylactically treated with OLE in which the delayed
mortalities also appeared but the percentage of mortalities is higher than post-infection
treated groups.
From these results it could be concluded that, OLE can be used as antiviral agent against
highly pathogenic H5N1 infection either as prophylactic and/or treatment therapy.
Further studies are needed to adjust the dose based on the mode of action of both extracts.
Key words: olive leaf extract, Antiviral, H5N1
Introduction
Infection of domestic poultry by AI viruses typically produces syndromes ranging from
asymptomatic infection to respiratory disease and drops in egg production to severe,
systemic disease with near 100% mortality (Easterday et al., 1997).
The AI virus belongs to the Orthomyxoviridae family of segmented negative-sense RNA
viruses All infl uenza viruses have eight different gene segments that encode at least 10
different viral proteins. The structural proteins in the mature virion can be divided into
the surface proteins that include the HA, neuraminidase (NA), and membrane ion channel
(M2) proteins and the internal proteins, including the nucleoprotein (NP), the matrix
protein (M1), and the polymerase complex composed of the polymerase basic protein 1
(PB1), polymerase basic protein 2 (PB2), and polymerase acidic protein (PA). Two
additional proteins produced by infl uenza viruses are the nonstructural proteins,
nonstructural protein 1 (NS1) and nonstructural protein 2 (NS2), which is also known as
the nuclear export protein (NEP) ( cited in Avian Influenza Edited by David E. Swayne
© 2008 John Wiley & Sons, Inc. ISBN: 978-0-813-82047-7
Clinical manifestations vary depending on the extent of damage to specific organs and
tissues In most cases in chickens and turkeys, the disease is fulminating with some birds
being found dead prior to observation of any clinical signs. If the disease is less
fulminating and birds survive for 3–7 days, individual birds may exhibit nervous
disorders such as tremors of head and neck, inability to stand, torticollis, opisthotonus,
and other unusual positions of head and appendages. (D. E. Swayne and D. A.
Halvorson, Avian influenza, chapter 6 p 153-184, diseases of poultry 12th edidtion
2008 , editor in chief Y.M.Saif)
Milder forms of AI were first recognized in various domestic poultry species between
1949 and the mid-1960s and have been termed low pathogenic, pathogenic, non-highly
pathogenic and low pathogenicity AI. Their impact on poultry production and trade has
been much less severe than with HPAI. (Alexander, 1987, Easterday and Tumova, 1978
and Senne et al., 1986)
In Egypt, In mid-February, 2006 devastating highly pathogenic avian influenza (HPAI)
H5N1 infected commercial poultry production sectors (Aly et al., 2006 a) and backyards
in Egypt (Aly et al., 2006 b). The outbreak caused great socioeconomic losses in poultry
industry (Kilany, 2006 and Safwat, 2006).
Controlling of the disease is only achieved by vaccination .The protection achieved by
different types of AI vaccine used is still under the hope for complete eradication of the
disease .
In diseased birds, the virus does not affected by the traditional antibiotics, so the
research for antiviral activity by herbal plants is considered a great hope for
controlling influenza viruses affecting all animal species
In another study done by Sylvia Lee-Huang et al., (2003) it revealed that OLE
has anti-HIV-1 activity. And they found that OLE inhibits acute infection and cell-to-cell
transmission of HIV-1 as assayed by syncytia formation using uninfected MT2 cells co-
cultured with HIV-1-infected H9 T lymphocytes. OLE also inhibits HIV-1 replication as
assayed by p24 expression in infected H9 cells. These anti-HIV effects of OLE are dose
dependent, with EC50s of around 0.2 µg/ml. There has been one anecdotal report
(Walker, 1996) that OLE augments the activity of the HIV-RT inhibitor 3TC.
AIDS patients have begun to use OLE for a variety of indications, among them to
strengthen the immune system, to relieve chronic fatigue, to boost the effects of anti-HIV
medications, and to treat HIV-associated Kaposi's sarcoma and HSV infections (Sylvia
Lee-Huang et al., 2003)
In this study, in-vitro and in-vivo assays are done to explore the antiviral effect of
OLE against H5N1 Influenza virus A/chicken/Egypt/1055/2010 (H5N1) (NLQP)
2- Material and methods
2.1 Materials & Methods
2.1.1 Extraction of OLE
Olive leaf extract: watery extract was Prepared according to Sylvia Lee-Huang et al.,
(2003). The leaves were ground into small pieces and extracted twice with sterile distilled
water for 12 h at 80°C, at a ratio of 40ml water to 1 g leaf. While the alcoholic extract
was kindly obtained from Department of pharmacognosy, faculty of Pharmacy, Cairo
University
2.2.2cell lines: Confluent monolayer of Madin-Darby Canine Kidney cells (MDCK) were
grown in T-25 tissue culture flasks
Avian Influenza Virus (AIV): A/chicken/Egypt/1055/2010 (H5N1) (NLQP), Accession
no. HQ198268
Chicken: a total of 50 four weeks old SPF chickens are devided into 5 groups each of 10
birds are used for protection test.
Cytotoxicity Assay:
It was done to determine the safest and highest tolerated dose (extract concentration)
causing no or minimal morphological changes (CPE) compared with control MDCK
cultures to be used further in the antiviral assays. The protocol was described in details by
Repetto et al, (2008)
Antiviral assays:
CPE Inhibition assay:
The assay was guided by Hu and Hsiung (1989), Vijayan et al. (2004) and
Elkosy et al. (2005)
Four Trials are designed as (A, B, C & D) : Trial A: MDCK cells infected with
H5N1 virus then treated with OLE after one hour of incubation, Trial B: MDCK cells
treated with OLE then infected with H5N1 virus after one hour of incubation, Trial C:
MDCK cells infected with H5N1 virus and treated with OLE at the same time
(competitive assay), Trial D: MDCK cells treated with a mixture of H5N1 virus and OLE
incubated for one hour at 37°C
Plaque reduction assay:
The plaque reduction assays were carried out as described earlier (Schmidtke et
al., 1998).and according to Ralph Tripp Lab protocols, 2005.
Invivo antiviral effect of OLE:
Five groups of SPF chicks each of 10 chicks are reared and named (G1, G2, G3, G4
& G5): G1: Treated group with OLE pre- and post infection with H5N1 virus, G2:
Treated group with OLE post-infection only , G3: Treated group with OLE pre-infection
only , G4: Non treated group with OLE ( only infected with H5N1). And G5: -ve control
group (not infected with AIV, not treated with OLE)
- Infection is done on 30 days of age with 0.2 ml intranasal of virus titer of 106 / ml
- The birds are observed after infection for symptoms and deaths
Results & Discussion
Olive leaf extract have potent antiviral activities against herpes mononucleosis,
hepatitis virus, rotavirus, bovine rhinovirus, canine parvovirus, and feline leukaemia virus
(Fredrickson, 2000). In addition, OLE has also exhibited a significant antiviral activity
against respiratory syncytial virus and parainfluenza type 3 virus (Ma et al., 2001).
OLE exhibits antiviral activity against viral haemorrhagic septicaemia
rhabdovirus (VHSV) and can be considered a potential source of antiviral agents for
aquaculture, which could lead to a new generation of chemicals with negligible
environmental impact to be safely administered to the aquatic media (Micol et al., 2005).
OLE inhibits the in vitro infectivity of ILT virus, and incubation of virus with the
extract before infection reduced the viral infectivity. Furthermore, it drastically decreased
the virus titer in a dose dependent manner when added to chicken embryo rough cell
(CER) 36 hours post-infection Kawther Zaher (2007).
The researchers (www.oliveleafextract.com) credit a number of unique properties
possessed by the olive leaf compound for the broad activity: an ability to interfere with
critical amino acid production essential for viruses, an ability to contain viral infection
and/or spread by inactivating viruses or by preventing virus shedding, budding or
assembly at the cell membrane, the ability to directly penetrate infected cells and stop
viral replication. In the case of retroviruses, it is able to neutralize the production of
reverse transcriptase and protease. These enzymes are essential for a retrovirus, such as
HIV, to alter the RNA of a healthy cell, and It can stimulate phagocytosis, an immune
system response in which cells ingest harmful microorganisms and foreign matter.
The existence of a vast reservoir of influenza A viruses in wild waterfowl and
shorebirds and the ability of these agents to jump species barriers means that they cannot
be eradicated and will always pose a threat to the health of humans and domestic animals.
the emergence of the virulent H5N1 virus has forced the world to approach it with the
seriousness it deserves.
Olive leaf extract has been claimed in a U.S. patent to have potent antiviral
activities against herpes mononucleosis, hepatitis virus, rotavirus, bovine rhinovirus,
canine parvovirus, and feline leukaemia virus (Fredrickson, 2000). In addition, OLE has
also exhibited a significant antiviral activity against respiratory syncytial virus and
parainfluenza type 3 virus (Ma et al., 2001).
Kawther Zaher (2007) found that OLE inhibits the in vitro infectivity of ILT
virus, and incubation of virus with the extract before infection reduced the viral
infectivity. Furthermore, it drastically decreased the virus titer in a dose dependent
manner when added to chicken embryo rough cell (CER) 36 hours post-infection. She
found also that, OLE is able to inhibite cell-to-cell membrane fusion induced by ILT
virus in uninfected cells, suggesting interactions with viral envelope.
Firstly, there is no available data regarding the effect of OLE on Avian influenza
virus to be compared with our results. The obtained results of antiviral (anti-cytopathic)
assays (table 1) revealed that treated MDCK cells prophylacticaly (Trail B) with OLE
either watery or alcoholic showing no CPE in comparison to the non treated cells.
While TCID only decreased by 1 and 2 logs in cells infected by AI then treated
after one hour with watery and Alcoholic OLE (Trail A)
In the competitive assay when the viral infection and OLE treatment are done at
the same time (Trail C), TCID significantly lowered (10-8.68 &10-7.87) for both watery and
alcoholic extracts respectively in comparison to non treated cells (10-14.25)
And in other trial to ( Trail D ) detect the direct effect of the extracts on the virus
itself, the incubated mixture of them for one hour is used and the results obtained
revealed that the virus titre decreased by 2 logs only in comparison to the non treated
cells.
Data obtained from plaque reduction assay (Table 2) also showing mostly the
same results. Briefly; Trail (B) in which the MDCK cells were treated with OLE then
infected with H5N1 virus after one hour of incubation showed decrease in plaque
forming unit by about (3.5 & 3) logs 10 PFU/ml. in watery and alcoholic extracts
respectively in comparison to the non treated cell culture and infected only with H5N1
(4.5x106 PFU/ml).
While Trial (A), showing no difference in PFU count when the cell were infected
with the virus and incubated for 1 hr then treated with the extracts.
Trial (C) where the viral infection and OLE treatment were done at the same time,
the virus titer was decreased by 2.5 and 1.5 logs as it reached 2x106 & 3x106 PFU/ml in
case of watery & alcoholic extracts respectively in comparison to the non treated cell
culture and infected only with H5N1 (4.5x106 PFU/ml). While in Trial (D) when the
virus–extract mix had been incubated for 1 hr before addition to the MDCK cell line,
only 1 log difference was noticed between treated and non treated cells.
These results suggest the prophylactic and ant-infective properties of the OLE rather
than the curative one, and the watery extract is more effective than the alcoholic one.
The watery extract is then used for the In-vivo study to determine the protective
effects of OLE against challenge of 30 days old chickens with H5N1 virus.
The results are shown in tables 3 & 4 and Figure (1), and it is clearly evidenced that
the OLE protect the birds 3 times more the negative control birds.
The use of OLE 3 days pre- and post infection protect 70 % of birds ( Group 1) while
group 2 in which the OLE is only given post infection also protect 70 % of birds. But the
difference appeared clinically by the delayed mortalities in case of pre-infection
treatment. This clinical result supported by the results of group C in which the birds
prophylacticaly treated with OLE in which has also delayed mortalities with higher
percentage than post-infection treated groups.
From these results it could be concluded that, OLE could be used as antiviral agent
against H5N1 infection either as prophylactic and/or treatment. The prophylactic therapy
appeared clinically to decrease the strength of signs and to delay the onset of mortalities.
For more accepted protecting effect the OLE could be used as 3 days pre- and 3 days
post-infection to both minimizing the symptoms and save more birds from mortalities so ,
it will be reflected more economically in poultry production
Also results confirmed that the OLE antiviral effect was not due to its direct effect on
the virion but due to the effect on the host cells by preventing or delaying cell to cell
transmission as agreed with Sylvia Lee-Huang et al., (2003)
We recommend more molecular investigations to confirm the actual mechanism of
OLE on the host cell. More work is also needed on the human level also to detect its
usefulness as a new antiviral agent for human against HPAI-H5N1 especially with the
emergence of resistance stains against the available medications.
Table (1): Anti-viral (anti-cytopathic) effect represented as TCID50:
Treatments
A
B
C
D
Virus only
10-14.25
10-14.25
10-14.25
10-14.25
Virus + OLE (watery)
10-13.135
No CPE
10-8.68
10-12.67
Virus + OLE (alcoholic)
10-12.7
No CPE
10-7.87
10-12.26
A: MDCK cells infected with H5N1 virus then treated with OLE after one hour of incubation
B: MDCK cells treated with OLE then infected with H5N1 virus after one hour of incubation
C: MDCK cells infected with H5N1 virus and treated with OLE at the same time (competitive assay)
D: MDCK cells treated with a mixture of H5N1 virus and OLE incubated for one hour at 37°C
Table (2): Plaque reduction assay represented as PFU/ml :
Treatments
A
B
C
D
Virus only
4.5x106
4.5x106
4.5x106
4.5x106
Virus + OLE (watery)
4.5x106
1x106
2x106
3x106
Virus + OLE (alcoholic)
5x106
1.5x106
3x106
3.5x106
Table (3): Invivo antiviral assay:
Group
Number of dead birds/ day
Mortality
Rate
2nd dpi*
3rd
4th
5th
6th
7th
8th
9th
10th
G1
-
2
-
-
1
-
-
-
-
30 %
G2
1
1
-
-
-
-
-
-
1
30 %
G3
-
2
1
1
-
-
-
-
-
40 %
G4
2
2
4
-
-
-
-
-
-
80 %
G5
-
-
-
-
-
-
-
-
-
0%
G1: Treated group with OLE pre- and post infection with H5N1 virus
G2: Treated group with OLE post-infection only
G3: Treated group with OLE pre-infection only
G4: Non treated group with OLE ( only infected with H5N1)
G5: -ve control group (not infected with AIV, not treated with OLE)
Table (4 ) : Protection % of OLE against the infection with H5N1:
Group
Protection %
G1
70 %
G2
70 %
G3
60 %
G4
G5
20 %
100%
References:
Alexander, D. J. 1987: Criteria for the definition of pathogenicity of avian influenza viruses. In
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150 words abstract:
Cytopathic inhibition assay was done for 4 trials; Results revealed that the treated MDCK
cells with either watery or alcoholic OLE showed no CPE in comparison to the non-
treated cells. Viral titer decreased by 1 to 2 logs in cells infected by AIV then treated with
either watery and/or Alcoholic OLE. In competitive assay, TCID was significantly
lowered in both watery and alcoholic extracts in comparison to non-treated cells. The
incubated mixture for one hour was used and the results obtained revealed that the virus
titer decreased by 2 logs only in comparison to the non-treated cells.
Plaque reduction assay showed the same results.
In vivo work, the use of OLE 3days pre- and 3 days post-infection protects 70 % of birds
with delayed beginning of mortalities in case of pre-infection treatment.
OLE can be used as antiviral agent against highly pathogenic H5N1 infection either as
prophylactic and/or treatment therapy.
