Anti-H5N1 virus new diglyceride ester from the Red Sea grass Thallasodendron ciliatum

Article abstract of DOI:10.1080/14786419.2012.742082

Some Egyptian plants were screened against highly pathogenic avian influenza strain H5N1 using plaque inhibition assay in Madin-Darby canine kidney. The results indicated that the extracts of Red Sea grass Thallasodendron ciliatum possessed potent antiviral activity (100% inhibition at the concentration of 1mgmL21). The bioactivityguided fractionations led to the isolation of a new diglyceride ester (1) along with asebotin (2) for the first time from the plant. The two isolates showed reduction of virus titre by 67.26% and 53.81% inhibition at concentration of 1 ngmL21, respectively.

Full text of DOI:10.1080/14786419.2012.742082

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Anti-H5N1 virus new diglyceride
ester from the Red Sea grass
Thallasodendron ciliatum
Amany K. Ibrahim a , Ahmed I. Youssef b , Abdel Satar Arafa c
,
Reda Foad a , Mohamed M. Radwan d , Samir Ross d e , Hashim A.
Hassanean a & Safwat A. Ahmed a
a Department of Pharmacognosy, Faculty of Pharmacy, Suez Canal
University, 41522, Ismailia, Egypt
b Division of Zoonoses, Department of Hygiene and Zoonoses,
Faculty of Veterinary Medicine, Suez Canal University, Ismailia,
Egypt
c National Laboratory for Veterinary Quality Control on Poultry
Production (NLQP), Animal Health Research Institute, Dokki, Giza,
Egypt
d School of Pharmacy, National Center for Natural Products
Research, University of Mississippi, University, MS, 38677, USA
e Department of Pharmacognosy, School of Pharmacy, University of
Mississippi, University, MS, 38677, USA
Published online: 19 Nov 2012.
To cite this article: Amany K. Ibrahim , Ahmed I. Youssef , Abdel Satar Arafa , Reda Foad ,
Mohamed M. Radwan , Samir Ross , Hashim A. Hassanean & Safwat A. Ahmed (2012): Anti-H5N1
virus new diglyceride ester from the Red Sea grass Thallasodendron ciliatum , Natural Product
Research: Formerly Natural Product Letters, DOI:10.1080/14786419.2012.742082
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Natural Product Research
iFirst article, 2012, 1–8
Anti-H5N1 virus new diglyceride ester from the Red Sea grass
Thallasodendron ciliatum
Amany K. Ibrahima, Ahmed I. Youssefb, Abdel Satar Arafac, Reda Foada,
Mohamed M. Radwand, Samir Rossde, Hashim A. Hassaneana and Safwat A. Ahmeda*
aDepartment of Pharmacognosy, Faculty of Pharmacy, Suez Canal University, 41522 Ismailia, Egypt;
bDivision of Zoonoses, Department of Hygiene and Zoonoses, Faculty of Veterinary Medicine, Suez Canal
University, Ismailia, Egypt; National Laboratory for Veterinary Quality Control on Poultry Production
c
(NLQP), Animal Health Research Institute, Dokki, Giza, Egypt; dSchool of Pharmacy, National Center for
e
Natural Products Research, University of Mississippi, University, MS 38677, USA; Department of
Pharmacognosy, School of Pharmacy, University of Mississippi, University, MS 38677, USA
(Received 30 July 2012; final version received 23 September 2012)
Some Egyptian plants were screened against highly pathogenic avian influenza strain
H5N1 using plaque inhibition assay in Madin–Darby canine kidney. The results
indicated that the extracts of Red Sea grass Thallasodendron ciliatum possessed potent
antiviral activity (100% inhibition at the concentration of 1 mg mL21). The bioactivity-
guided fractionations led to the isolation of a new diglyceride ester (1) along with
asebotin (2) for the first time from the plant. The two isolates showed reduction of virus
titre by 67.26% and 53.81% inhibition at concentration of 1 ng mL21, respectively.
Keywords: Thallasodendron ciliatum; diglyceride ester; asebotin; anti-influenza virus
(H5N1)
1. Introduction
Influenza A viruses belong to the family Orthomyxoviridae and have been isolated from a
variety of different species. Further subtyping of influenza A viruses is based on antigenic
differences between the two surface glycoproteins haemagglutinin (H1–H16) and
neuraminidase (N1–N9) of the influenza A viruses (Fouchier et al., 2005). Of them, human
H1N1 and H3N2 influenza A viruses are highly contagious and are the main cause of ‘seasonal
influenza’ worldwide (Fiore, Shay, Broder, Iskander, & Uyeki, 2008). Since late 2003, highly
pathogenic avian influenza (HPAI) H5N1 viruses have appeared in Southeast Asian countries,
and have become a global problem with human involvement (Hien, De Jong, & Farrar, 2004).
Recently, in 2009, swine–origin pandemic (H1N1) virus has emerged and spread rapidly all
over the world (Dawood, Jain, Finelli, Shaw, & Lindstrom, 2009). In mid-February 2006, a
devastating HPAI H5N1 virus disease has become enzootic in poultry throughout Egypt and still
circulates in the poultry population (Aly, Arafa, & Hassan, 2008). In that period, Egypt had the
highest number of human avian influenza cases outside Asia, and the second highest number
worldwide. Until now human avian influenza remains as unresolved threat to veterinary and
public health with zoonotic transmission potential to human in Egypt (Kandeel et al., 2006).
*Corresponding author. Email: safwat_aa@yahoo.com
ISSN 1478-6419 print/ISSN 1478-6427 online
q 2012 Taylor & Francis
http://dx.doi.org/10.1080/14786419.2012.742082
http://www.tandfonline.com

2
A.K. Ibrahim et al.
The application of antiviral drugs during the early phase of a pandemic could be of help to
control it (Monto, 2005). Detection of new, potent and cheap anti-influenza agents from plant
and marine extracts could be used for the treatment and prevention of influenza, particularly for
HPAI H5N1 strain, for avian population which, in turn, will prevent zoonotic transmissions and
will limit the spread of the viruses.
Currently, two types of anti-influenza drugs are available: M2 ion channel blockers (amino-
adamantines: amantadine and rimantadine) and neuraminidase inhibitors (oseltamivir and
zanamivir) (Davies et al., 1994; Hayden, Cote, & Douglas, 1980). However, amino-adamantine-
resistant viruses readily emerged and are already prevalent worldwide among the seasonal influenza
viruses (Bright, Medina, Xu, Perez-Oronoz, & Wallis, 2005; Bright, Shay, Shu, Cox, & Klimov,
2006). Oseltamivir-resistant H5N1 viruses have been isolated from patients (De Jong, Tran,
Truong, Vo, & Smith, 2005). In addition, current production capability has limitations and is
insufficient to provide adequate amounts of drugs for mass treatment and chemoprophylaxis.
Current medicines are always dogged with anxiety for emergence of resistant viruses. Thus, in the
advent of an influenza virus pandemic, it is likely that the administration of antiviral drugs will be an
important first line of defense against the virus. Therefore, we try to find new drugs with anti-
influenza virus activity through the evaluation of this activity in certain Egyptian natural extracts.
The Egyptian flora and Red Sea are rich in plants that have been used by the local
communities for the treatment of a large number of ailments. Therefore, plants of Egypt still
need more investigation to detect their importance in the discovery of new sources for new
molecules that can be used in the treatment of different ailments following their traditional uses.
Screening of different Egyptian plants against influenza virus (H5N1) led to the discovery of Red
Sea grass Thallasodendron ciliatum, which showed potent activity against H5N1 influenza virus.
Sea grasses are monocotyledonous vascular flowering plants that live in coastal and estuarine
areas of the world. They are unique in that they are usually totally submerged in the water, they
possess a root system with stems buried within a soft substrate, they have vegetative and sexual
reproduction and have flowers fertilised by water-borne pollen (Aioi, Mukai, & Koike, 1981).
There are approximately 50 species of sea grasses in 12 genera. Seven genera are considered
tropical, while the remaining five are more or less confined to temperate waters (Pollard &
Greenway, 1993). Sea grass can be found all over the world except in the polar region. In many
places, sea grasses cover extensive areas, which are usually called as sea grass beds. Of the types
of communities that make up single sea grass beds, is T. ciliatum (Phillips & Durako, 2000).
T. ciliatum is a tropical sea grass which can be classified as sub-tidal and not deep water bed-
forming sea grass (Carruthers et al., 2001).
The anti-influenza H5N1-guided fractionation led to the isolation and structure elucidation
of a new metabolite diglyceride ester (1) along with the previously known metabolite, asebotin
(2), for the first time from T. ciliatum. The two isolates showed reduction of virus titre by 67.26%
and 53.81% at concentration of 1 ng mL21, respectively.
2. Results and discussion
Based on the acquired potent antiviral activity (100% inhibition at the concentration of
1 mg mL21) of the T. ciliatum extract, the bioactivity-guided chromatographical processing was
run. Two compounds were purely isolated and spectroscopically analysed. The structure
elucidation procedure of the two compounds would be stated in the following.
2.1. Compound 1
The HR-ESI-MS spectrum of 1 displayed a pseudo-molecular ion peak at m/z 619.5298 [MþH]þ
which when combined with detailed analysis of the 13C spectrum and Distortionless

Natural Product Research
3
Enhancement by Polarization Transfer (DEPT) indicated a molecular formula of C39H70O5
representing five units of unsaturation. Analysis of 13C NMR spectrum and DEPT indicated the
presence of two oxygenated methylenes at d 62.1, one oxygenated methine at d 68.9 together
with two CO for the two ester moieties at d 172.8 and 173.2. Also the presence of three di-
substituted olefinic moieties was confirmed by 13C NMR resonances at d 127.8, 128.0, 129.6,
129.8, 129.9 and 130.1. The above-mentioned findings were consistent with the presence of a
diglyceride ester. GC–MS analysis of the fatty acid methyl ester of 1 was carried out after
hydrolysis and yielded two peaks with molecular ions of m/z (326) and (264) corresponding to
(C16:3) and C20 fatty acid methyl esters. The fatty acids were identified as the unsaturated
fatty acids; (4E,7E,10E)-hexadeca-4,7,10-trienoic acid and arachidic acid by library search
National Institute of Standards and Technology (NIST) and by comparing with published data
(Asselineau & Lededrer, 1961; Xu, Miao, & Wu, 2006). The placement of the fatty acid at C-1
and C-3 of the diglyceride ester was assigned by 1H–1H COSY between H-2 and H2-1 and H-2
and H2-3. This was confirmed by HMBC of H2-1/C-2 (2JCH), H2-1/C-3 (3JCH), H2-1/C-10 (3JCH),
H2-3/C-2 (2JCH), H2-3/C-1 (3JCH), H2-3/C-100 (3JCH), H-2/C-1 (2JCH), H-2/C-3 (2JCH) which
revealed that 1 is 1,3-diglyceride ester (Figure 1). The position of the double bonds was
confirmed by 1H–1H COSY spectrum between H-400/H-500, H-500/H2-600, H2-600/H-700, H-700/H-800,
H-800/H2-900 and H2-900/H-1000 and also from HMBC of H2-200/C-300 (2JCH), H2-200/C-400 (3JCH), H2-
300/C-500 (3JCH), H2-600/C-800 (3JCH), H2900/C-1100 (3JCH) and H2-1200/C-10 (3JCH), leading to the
assignment of the C-400/C-500/C-600/C-700/C-800/C-900/C-1000/C-1100/C-1200 (Figure 1). Detailed
analysis of COSY and HMBC correlations was found to be in complete agreement with the
proposed structure for 1. The name 3-(4E,7E,10E-hexadeca-4,7,10-trienoyloxy)-2-hydroxypro-
pyl icosanoate was assigned to the new diglyceride.
2.2. Compound 2
This was identified as asebotin by comparing its spectroscopic properties with those reported in
the literature (Yao et al., 2005).
O
9'
1
20'
H2C O
1'
10'
2CH
OH
10''
1''
H2C O
16''
4''
3
7''
O
1
OH
H3CO
O
OH
OH
O
O
HO
OH
OH
1
2
O
20'
H2C O
HC OH
2
H2C O
16''
3
O
Figure 1. Important HMBC (arrows) and COSY (bold) correlations of 1.

4
A.K. Ibrahim et al.
2.3. Anti-influenza activity
Anti-influenza activity of dilutions of the extract, its fractions and the pure compounds was
measured by a plaque inhibition assay in Madin–Darby canine kidney (MDCK). The extract
completely inhibited the growth of the virus at the concentration of 1 mg mL21 as compared to the
antiviral drug zanamivir, whereas compounds 1 and 2 showed reduction of virus titre by 67.26%
and 53.81% inhibition at concentration of 1 ng mL21, respectively (Table 1 and Figure 2).
3. Materials and methods
3.1. General experimental procedures
1H NMR (400 MHz), 13C NMR (100 MHz), DEPT-135 and 2D-NMR spectra were recorded
using the residual solvent signal as an internal standard on a Varian AS 400 (Varian Inc., Palo
Alto, CA, USA). IR spectra were recorded on a Bruker Tensor 27 (Bruker Corporation, Billerica,
MA, USA). UV spectra were obtained on a Varian Cary 50 Bio UV–visible spectrophotometer.
The UV spectra were recorded on a double beam Shimadzu UV–visible spectrophotometer
(model UV-1601 PC, Kyoto City, Japan). IR spectra were recorded on Nicolit FT IR
spectrophotometer (Nicolet Company, Nicolet, Canada) range 400–4000, USA. High resolution
mass spectra were recorded using a Bruker BioApex (Bruker Corporation).
Fatty acid methyl esters were identified using Hewlett Packard (HP) gas liquid
chromatography, series 6890 equipped with flame ionisation detector. A capillary column
(HP-INNOWAX, polyethylene glycol, 30 m £ 530 mm, film thickness 1.00 mm) was used in
separation of fatty acids. The injector port temperature was set at 2508C (splitless mode) and a
pressure of 14.81 psi and the detector cell at 2758C. The flow rate of the carrier gas (N2) was
30 mL/min. The initial column temperature was 708C and increased to 2008C by the rate of 48C/
min, then isothermally for a total run time of 32.5 min.
Pre-coated silica gel G-25 UV254 plates were used for thin layer chromatography (TLC)
(20 cm £ 20 cm) (E. Merck, Darmstadt, Germany). Silica gel Purasil 60A, 230–400 mesh was
used for flash column chromatography (Whatman, Sanford, ME, USA).
3.2. Plant material, collection and identification
The sea grass T. ciliatum (coll. no. SAA-41) was collected from Safaga at the Egyptian Red Sea,
air-dried and stored at low temperature (2248C) until processed. The plant was identified by
Table 1. In vitro anti-influenza virus activity of T. ciliatum extract, its fraction and compounds 1 and 2.
Test extracts and compounds
Concentration
Plaque assay (mean ^ SD)
Plaque inhibition (%)
Virus control
Zanamivir
Extract
Hexane
25% AcOEt in Hexane
50% AcOEt in Hexane
75% AcOEt in Hexane
AcOEt
25% MeOH in AcOEt
50% MeOH in AcOEt
75% MeOH in AcOEt
MeOH
22.3 ^ 1.52
10 ng mL21
1 mg mL21
1 mg mL21
1 mg mL21
1 mg mL21
1 mg mL21
1 mg mL21
1 mg mL21
1 mg mL21
1 mg mL21
1 mg mL21
1 ng mL21
1 ng mL21
0
0
100
100
0
37.3
0
0
0
31.3
0
0
14 ^ 1
15.3 ^ 0.6
0
Compound 1
Compound 2
7.33 ^ 1.52
10.33 ^ 1.15
67.26
53.81

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5
Figure 2. The antiviral effect of compounds (1 and 2) and their related extracts against H5N1 virus
compared to zamivir using plaque inhibition assay in MDCK.
Dr Tarek Temraz, Marine Science Department, Faculty of Science, Suez Canal University,
Ismailia, Egypt. A voucher specimen was deposited in the herbarium section of Pharmacognosy
Department, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt under registration
number SAA-41.
3.3. Extraction and isolation
T. ciliatum was dried (700 g dry weight), grounded and extracted with a mixture of MeOH/
CH2Cl2 (1:1) (3 £ 2 L) at room temperature. The extract was evaporated under vacuum to afford
150 g residue. The residue was subjected to vacuum liquid chromatography on a flash silica gel
using hexane, EtOAc and MeOH gradient.
The fraction eluted using 25% EtOAc in hexane was concentrated to afford 5 g of green
residue. Purification of this fraction was done by flash column chromatography on silica gel
using hexane/EtOAc (95:5). Fractions with the same TLC pattern were combined and
rechromatographed by flash column chromatography on silica gel using hexane/EtOAc (95:5) to
afford 1 (156 mg) (Rf ¼ 0.70, 10% EtOAc/hexane).
The fraction eluted with 25% MeOH in EtOAc was concentrated to afford 4 g residue, which
was purified by column chromatography on silica gel using hexane/EtOAc (10:90) to MeOH/
EtOAc (25:75). Fractions eluted with 5% MeOH in EtOAc with the same TLC pattern were
combined and rechromatographed by flash column chromatography on silica gel using 5%
MeOH in EtOAc. Final purification was done by crystallisation from MeOH to afford 2 (8 mg)
(Rf ¼ 0.50, 10% MeOH/CHCl3).
3.4. Hydrolysis of 1
Compound 1 was refluxed with 10% ethanolic KOH and benzene for 24 h. After adding H2O, the
reaction mixture was extracted with ether. The aqueous alkaline solution was acidified with HCl
and extracted with ether. The ether fraction was concentrated to yield a fatty acid.

6
A.K. Ibrahim et al.
3.5. Preparation of fatty acid methyl esters of 1
Prepared fatty acid of 1 was refluxed with MeOH and concentrated H2SO4 for 1 h. After adding
H2O, the reaction mixture was extracted with ether and dried over anhydrous MgSO4. The
resulting ether-soluble fraction was analysed by GC–MS.
3.6. Isolates
Compound (1): colourless oil, Rf ¼ 0.70, 10% EtOAc/hexane; UV (MeOH) lmax (log 1)
219 nm; IR (KBr) (thin film) nmax 3506.23, 2130.56, 1655.90, 1432.39, 1031.21, 955.28 cm21
;
1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz). HR-ESI-MS (positive ion mode)
1
m/z ¼ 619.5298 [M þ H]þ (calcd for C39H70O5:619.5302). H NMR (CDCl3, 400 MHz): dH
0.87 (6H, t, J ¼ 6.8 Hz, H3-1600 and H3-200), 1.24 (44H, m, H2-40-190 and H2-1300-1500), 1.59 (2H,
m, H2-30), 1.99 (6H, m, H2-600, H2-900 and H2-120), 2.02 (2H, m, H2-300), 2.31 (4H, m, H2-20 and
H2-200), 2.78 (2H, m, H2-600), 4.17 (2H, dd, J ¼ 2.4, 6.8 Hz, H-1 and H-3), 4.29 (2H, dd, J ¼ 2.4,
6.8 Hz, H-1 and H-3), 5.20 (H, q, J ¼ 1.6 Hz, H-2), 5.33 (6H, m, H-400, H-500, H-700, H-800, H-1000
and H-1100), 13C NMR (CDCl3, 100 MHz): dC 14.1 (C-200), 14.2 (C-1600), 22.5 (C-190), 24.8 (C-
1500), 24.9 (C-30), 25.6 (C-600 and C-900), 27.2 (C-300), 29.1 (C-1400), 29.6 (C-40-180 and C-1300),
31.8 (C-1200), 31.9 (C-60 and C-90), 33.9 (C-20), 34.2 (C-200), 62.1 (C-1 and C-3), 68.9 (C-2),
127.8 (C-1000), 128.0 (C-500), 129.6 (C-700), 129.8 (C-800), 129.9 (C-400), 130.1 (C-1100), 172.8 (10),
173.2 (100).
3.7. Biological activity
3.7.1. Anti-influenza virus activity
Cells and viruses.
Virus. HPAI strain H5N1 from Egypt (field strain).
Strain. A/chicken/Egypt/1055/2010(H5N1), Accession number: HQ198268.
Virus titre. TCID50 ¼ 107 mL21 and 22 PFU mL21 (plaque forming unit).
Virus propagation and titration.
(a) Propagation of the viruses in cell culture
Viruses were propagated in MDCK cells, and their culture supernatants were used as virus
stocks. The stocks were titrated in MDCK cells and stored at 2808C until use. The virus
titre was expressed as PFU mL21. MDCK cells were grown in minimal essential
medium (MEM) containing 10% foetal calf serum 4 mM-glutamine and antibiotics, and
maintained in MEM supplement with 5% newborn calf serum, 4 mM L-glutamine and
antibiotics. All cells were maintained at 378C and 5% CO2.
(b) Propagation of the viruses in embryonated chicken eggs
Embryonated hens’ eggs were incubated horizontally at 37.78C and a relative humidity
of 50–60% under forced air circulation. From the day 3 after starting incubation, the
eggs were automatically turned by 180 on their axis every 4 h. Before inoculation on
day 11, the eggs were candled to eliminate infertile eggs and dead embryos. For
influenza virus isolation in ovo, 0.1 mL of the sample’s phosphate-buffered saline
(PBS) solution suspension was dropped on the chorioalantoic (CA) cavity of 11-day
embryonated eggs by using a tuberculin syringe fitted with a 27-gauge needle. PBS
was control injected into the CA cavity of the egg as control negative; also, positive
control was included. Inoculated embryos were incubated at 378C for 24–48 h. Virus
isolation was confirmed by conventional and real-time PCR techniques.

Natural Product Research
7
3.7.2. Cytotoxicity evaluation
The evaluation of cytotoxicity of extracts was done at a final concentration of 1 mg mL21, while
for pure compounds at a concentration of 1 ng mL21. The extract and its fractions were assessed
in standard thiazolyl blue tetrazolium bromide (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide, MTT) assay following 24-h incubation with cells (Mosmann, 1983).
3.7.3. Anti-influenza evaluation
The anti-influenza activity of dilutions of the extract, its fractions and the pure compounds was
measured by a plaque inhibition assay in MDCK as previously described (Hayden, 2001). Briefly,
confluent monolayer of MDCK cells in six-well plates was infected with the virus at a concentration
of 22 £ 1026 PFU mL21 or at a multiplicity of infection of 0.1. After 1-h incubation, the inocula
were removed and the cells were washed twice with PBS. The cells were then overlaid with a
1% agarose-MEM and a designated concentration of each extract for 48h in a 5% CO2 incubator
at 378C. The cells were fixed with 10% neutral-buffered formalin, stained with crystal violet
and the plaque number was counted. Positive control (virus without compounds), negative control
(cells only) and zanamivir (obtained from GlaxoSmithKline, London, United Kingdom)
10ngmL21 were included.
The same experiment was carried out three times independently for each drug, and the plaque
number was calculated as the average ^ standard deviation (SD) of the three experiments.
4. Conclusions
In this study, some Egyptian plants and marine organisms (data not shown) were tested for their
in vitro activity against HPAI strain H5N1. Of the tested plants, T. ciliatum possessed potent
antiviral activity (100% inhibition at the concentration of 1 mg mL21) as compared to the antiviral
drug zanamivir. In favour of this promising extract activity, a bioassay-guided fractionation was
conducted using a gradient of EtOAc/hexane, then a gradient of MeOH/EtOAc. The antiviral
activity was found to be concentrated at 25% EtOAc in hexane and 25% MeOH in EtOAc
fractions in concentrations of 1 mg mL21 each. Based on these findings, further purification of the
active fractions was done to yield the two pure compounds 1 and 2. The activity of the extract, its
fractions and the pure compounds was measured by a plaque inhibition assay in MDCK, where
the plaque inhibition (%) of the pure compounds was found to be even more than that of their
parent fractions (Table 1). From the results given in Table 1, compounds 1 and 2 were found to
inhibit the virus with a concentration of 1 ng mL21 each by a percentage of 67.26 and 53.81,
respectively, which had a potency comparable to that of the clinically used antiviral drug
zanamivir which had a complete inhibition but at higher concentration of 10 ng mL21
.
Supplementary material
Supplementary Figures S1–S9 relating to this article are available online.
Acknowledgements
The authors are grateful to Dr Tarek Temraz, Faculty of Science, Suez Canal University for taxonomical
identification of the sea grass.
References
Aioi, K., Mukai, H., & Koike, I. (1981). Growth and or-ganic production of eelgrasss (Zostera marina L.) in temperate
waters of the Pacific coast of Japan. II. Growth analysis in winter. Aquatic Botany, 10, 175–182.

8
A.K. Ibrahim et al.
Aly, M.M., Arafa, A., & Hassan, M.K. (2008). Epidemiological findings of outbreaks of disease caused by highly
pathogenic H5N1 avian influenza virus in poultry in Egypt during 2006. Avian Diseases, 52(2), 269–277.
Asselineau, J., & Lededrer, E. (1961). Chemistry of lipids. Annual Review Biochemistry, 30, 71–92.
Bright, R.A., Medina, M.J., Xu, X., Perez-Oronoz, G., & Wallis, T.R. (2005). Incidence of adamantane resistance among
influenza A (H3N2) viruses isolated worldwide from 1994 to 2005: A cause for concern. Lancet, 366,
1175–1181.
Bright, R.A., Shay, D.K., Shu, B., Cox, N.J., & Klimov, A.I. (2006). Adamantane resistance among influenza A viruses
isolated early during the 2005–2006 influenza season in the United States. The Journal of the American Medical
Association, 295, 891–894.
Carruthers, T.J.B., Dennison, W.C., Longstaff, B.J., Waycott, M., Abal, E.G., McKenzie, L.J., & Long, W.J.L. (2001).
Seagrass habitats of north east Australia: Models of key processes and controls. Bulletin of Marine Science, 71,
1153–1169.
Davies, W.L., Grunert, R.R., Haff, R.F., McGahen, J.W., Neumayer, E.M., Paulshock, M., . . . , Hoffmann, C.E. (1994).
Antiviral activity of 1-adamantanamine (amantadine). Science, 144, 862–863.
Dawood, F.S., Jain, S., Finelli, L., Shaw, M.W., & Lindstrom, S. (2009). Emergence of a novel swine-origin influenza A
(H1N1) virus in humans. The New England Journal of Medicine, 360, 2605–2615.
De Jong, M.D., Tran, T.T., Truong, H.K., Vo, M.H., & Smith, G.J. (2005). Oseltamivir resistance during treatment of
influenza A (H5N1) infection. The New England Journal of Medicine, 353, 2667–2672.
Fiore, A.E., Shay, D.K., Broder, K., Iskander, J.K., & Uyeki, T.M. (2008). Prevention and control of influenza:
Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recommendations
and Reports, 57, 1–60.
Fouchier, R.A., Munster, V., Wallensten, A., Bestebroer, T.M., Herfst, S., Smith, D., . . . , Osterhaus, A.D. (2005).
Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls.
Journal of Virology, 79(5), 2814–2822.
Hayden, F.G. (2001). Perspectives on antiviral use during pandemic influenza. Philosophical Transactions of the Royal
Society of London Series B, Biological Sciences, 356, 1877–1884.
Hayden, F.G., Cote, K.M., & Douglas, Jr., R.G. (1980). Plaque inhibition assay for drug susceptibility testing of influenza
viruses. Antimicrobial Agents and Chemotherapy, 17(5), 865–870.
Hien, T., De Jong, M., & Farrar, J. (2004). Avian influenza: A challenge to global health care structures. The New
England Journal of Medicine, 351(23), 2363–2365.
Kandeel, A., Manoncourt, S., Abd el Kareem, E., Mohamed, A.N., El-Refaie, S., Essmat, H., . . . , El-Sayed, N. (2006).
Zoonotic transmission of avian influenza virus (H5N1), Egypt. Emerging Infectious Diseases, 16(7), 1101–1107.
Monto, A.S. (2005). The threat of an avian influenza pandemic. The New England Journal of Medicine, 352(4), 323–325.
Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: Application to proliferation and
cytotoxicity assays. Journal of Immunological Methods, 65, 55–63.
Phillips, R.C., & Durako, M.J. (2000). Global status of sea-grassses. Seas at the millenium: An environmental evaluation
(Vol. 3, pp. 1–16). Amsterdam: Elsevier.
Pollard, P.C., & Greenway, M. (1993). Photosynthetic char-acteristics of seagrassses, Cymodocea serrulata, Thalassia
hemprichii, and Zostera capricorni, in a low-light environment, with a comparison of leaf-marking and lacunal-
gas measurements of productivity. Australian Journal of Marine and Freshwater Research, 44, 127–139.
Xu, H., Miao, X., & Wu, Q. (2006). High quality biodiesel production from a microalga Chlorella protothecoides by
heterotrophic growth in fermenters. Journal of Biotechnology, 126, 499–507.
Yao, G., Ding, Y., Zuo, J., Wang, H., Wang, Y., Ding, B., . . . , Qin, G. (2005). Dihydrochalcones from the Leaves of
Pieris japonica. Journal of Natural Products, 68, 392–396.